Combined chemical and genetic approaches for generation of induced pluripotent stem cells

ABSTRACT

The present invention provides for identification and use of small molecules to induce pluripotency in mammalian cells as well as other methods of inducing pluripotency.

CROSS-REFERENCE TO RELATED PATENT APPLICATIONS

This application is a continuation of application Ser. No. 12/933,391,filed Jan. 4, 2011, which is a U.S. National Stage entry under 35 U.S.C.§371 of PCT/US2009/037429, filed Mar. 17, 2009, which claims benefit ofpriority to U.S. Provisional Patent Application Nos. 61/069,956, filedMar. 17, 2008 and 61/197,986, filed Oct. 31, 2008, each of which areincorporated by reference.

REFERENCE TO A SEQUENCE LISTING, A TABLE OR A COMPUTER PROGRAM LISTINGAPPENDIX SUBMITTED AS AN ASCII TEXT FILE

The Sequence Listing written in file 77103-769176_ST25.TXT, created onFeb. 11, 2015, 1,273 bytes, machine format IBM-PC, MS Windows operatingsystem, is hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Stem cells are often classified as totipotent or pluripotent. Atotipotent stem cell has differentiation potential which is total: itgives rise to all the different types of cells in the body. A fertilizedegg cell is an example of a totipotent stem cell. Pluripotent stem cellscan give rise to any cell type in the body derived from the three maingerm cell layers or an embryo itself.

Pluripotent stem cells, such as embryonic stem cells (ESCs), proliferaterapidly while maintaining pluripotency, namely, the ability todifferentiate into various types of cells. Embryonic stem cells arepromising donor sources for cell transplantation therapies. However,human ESCs are also associated with ethical issues regarding the use ofhuman embryos and rejection reactions after allogenic transplantation.It may be possible to overcome these issues by generating pluripotentstem cells directly from a patient's somatic cells. That somatic cellnuclei acquire an embryonic stem-like status by fusion with ESCssuggests the existence of ‘pluripotency-inducing’ factors. Previousstudies have recently shown that retrovirus-mediated transfection withfour transcription factors (Oct-3/4, Sox2, KLF4 and c-Myc), which arehighly expressed in ESCs, into mouse fibroblasts has resulted ingeneration of induced pluripotent stem (iPS) cells. See, Takahashi, K. &Yamanaka, S. Induction of pluripotent stem cells from mouse embryonicand adult fibroblast cultures by defined factors. Cell 126, 663-676(2006); Okita, K., Ichisaka, T. & Yamanaka, S. Generation ofgermline-competent induced pluripotent stem cells. Nature 448, 313-317(2007); Wernig, M. et al. In vitro reprogramming of fibroblasts into apluripotent ES-cell-like state. Nature 448, 318-324 (2007); Maherali, N.et al. Directly reprogrammed fibroblasts show global epigeneticremodeling and widespread tissue contribution. Cell Stem Cell 1, 55-70(2007); Meissner, A., Wernig, M. & Jaenisch, R. Direct reprogramming ofgenetically unmodified fibroblasts into pluripotent stem cells. NatureBiotechnol. 25, 1177-1181 (2007); Takahashi, K. et al. Induction ofpluripotent stem cells from adult human fibroblasts by defined factors.Cell 131, 861-872 (2007); Yu, J. et al. Induced pluripotent stem celllines derived from human somatic cells. Science 318, 1917-1920 (2007);Nakagawa, M. et al. Generation of induced pluripotent stem cells withoutMyc from mouse and human fibroblasts Nature Biotechnol. 26, 101-106(2007); Wernig, M., Meissner, A., Cassady, J. P. & Jaenisch, R. c-Myc isdispensable for direct reprogramming of mouse fibroblasts. Cell StemCell 2, 10-12 (2008). iPS cells are similar to ESCs in morphology,proliferation, and pluripotency, judged by teratoma formation andchimaera contribution.

A recent breakthrough of using defined genetic manipulation, i.e. viraltransduction of few genes highly and/or specifically expressed in mouseor human embryonic stem (ES) cells, in reprogramming both mouse andhuman somatic cells to induced pluripotent stem (iPS) cells has openedup tremendous opportunities to generate patient-specific stem cells forvarious applications (e.g. cell-based therapy or drug discovery) withoutthe controversies associated with the conventional human ES cells, aswell as to study the epigenetic reversal process. Ultimate clinicalapplication of an iPS-cell approach would largely require methods ofdirected differentiation of human PS cells for generating homogenouspopulations of lineage-specific cell types as well as eliminating risksassociated with the current iPS-cell drawbacks of genetic manipulationand low efficiency/slow kinetics. Recent studies have shown that one ofthe previously required four genes, cMyc, is dispensable foroverexpression in generating iPS cells. See, Nakagawa, M. et al.Generation of induced pluripotent stem cells without Myc from mouse andhuman fibroblasts Nature Biotechnol. 26, 101-106 (2007); Wernig, M.,Meissner, A., Cassady, J. P. & Jaenisch, R. c-Myc is dispensable fordirect reprogramming of mouse fibroblasts. Cell Stem Cell 2, 10-12(2008). However, the reprogramming efficiency was substantially reducedwith also much slower reprogramming kinetics in the absence of cMyc.

BRIEF SUMMARY OF THE INVENTION

The present invention provides methods for screening for agents thatinduce reprogramming or dedifferentiation of mammalian cells intopluripotent stem cells. In some embodiments, the method comprises,

a) introducing at least one of, but not all of, an Oct polypeptide, aKlf polypeptide, a Myc polypeptide, and a Sox polypeptide intonon-pluripotent cells to generate transfected cells;

b) contacting the transfected cells with a library of different agents;

c) screening the contacted cells for pluripotent stem cellcharacteristics; and

d) correlating the development of stem cell characteristics with aparticular agent from the library, thereby identifying an agent thatstimulates dedifferentiation of cells into pluripotent stem cells.

In some embodiments, step a) comprises introducing one or moreexpression cassettes for expression of the at least one of, but not allof, an Oct polypeptide, a Klf polypeptide, a Myc polypeptide, and a Soxpolypeptide into the non-pluripotent cells.

In some embodiments, step a) comprises introducing at least one of, butnot all of, an exogenous Oct polypeptide, an exogenous Klf polypeptide,an exogenous Myc polypeptide, and an exogenous Sox polypeptide into thenon-pluripotent cells.

In some embodiments, the particular agent is between 50-1500 daltons.

In some embodiments, step a) comprises introducing two expressioncassettes into the cells, wherein each expression cassette comprises apolynucleotide encoding a different protein, wherein the protein isselected from the group consisting of an Oct polypeptide, a Klfpolypeptide, a Myc polypeptide, and a Sox polypeptide, and the remainingmembers of the group are not introduced into the cells.

In some embodiments, step a) comprises introducing three expressioncassettes into the cells, wherein each expression cassette comprises apolynucleotide encoding a different protein, wherein the protein isselected from the group consisting of an Oct polypeptide, a Klfpolypeptide, a Myc polypeptide, and a Sox polypeptide, and the remainingmember of the group is not introduced into the cells.

In some embodiments, the cells are human cells. In some embodiments, thecells are non-human mammalian cells. In some embodiments, thenon-pluripotent cells are progenitor cells. In some embodiments, theprogenitor cells are neural progenitor cells, skin progenitor cells orhair follicle progenitor cells.

In some embodiments, the Oct polypeptide is Oct4, the Klf polypeptide isKlf 4, the Myc polypeptide is c-Myc, and the Sox polypeptide is Sox2.

The present invention also provides for methods of screening formammalian cells with pluripotent stem cell characteristics. In someembodiments, the method comprises

a) contacting cells with a MAPK/ERK kinase (MEK) inhibitor such thatgrowth of non-pluripotent cells is inhibited and growth of pluripotentstem cells is promoted; and

b) screening the contacted cells for pluripotent stem cellcharacteristics.

In some embodiments, the method comprises

contacting the cells with a library of agents prior to step a);

and, following step b), selecting an agent that induces pluripotent stemcell based on the results of step b).

In some embodiments, the cells are human cells. In some embodiments, thecells are mouse, dog, cow, pig, rat and non-human primate cells.

In some embodiments, the MEK inhibitor is PD0325901.

The present invention also provides methods of producing inducedpluripotent stem cells from mammalian non-pluripotent cells. In someembodiments, the method comprises,

a) introducing one or more of an Oct polypeptide, a Klf polypeptide, aMyc polypeptide, and a Sox polypeptide into the non-pluripotent cells;

b) contacting the cells with an agent that inhibits H3K9 methylation orpromotes H3K9 demethylation, thereby producing induced pluripotent stemcells.

In some embodiments, step a) comprises contacting the non-pluripotentcells with one or more exogenous polypeptides selected from a Klfpolypeptide, an Oct polypeptide, a Myc polypeptide, and a Soxpolypeptide. In some embodiments, step a) comprises at least two (e.g.,2, 3, 4, 5, or more) cycles of:

i. contacting the non-pluripotent cells with one or more exogenouspolypeptides selected from a Klf polypeptide, an Oct polypeptide, a Mycpolypeptide, and a Sox polypeptide;

ii. followed by culturing the cells in the absence of the exogenouspolypeptides.

In some embodiments, step a) comprises introducing one or moreexpression cassettes for expression of a Klf polypeptide, an Octpolypeptide, a Myc polypeptide, and a Sox polypeptide into thenon-pluripotent cells.

In some embodiments, the method further comprises screening thecontacted cells for pluripotent stem cell characteristics.

In some embodiments, an expression cassette for expression of an Octpolypeptide and an expression cassette for expression of a Soxpolypeptide are introduced into the non-pluripotent cells.

In some embodiments, the introducing step comprises introducing into thenon-pluripotent cells one or more expression cassettes for expression ofa KLF polypeptide and an Oct polypeptide, wherein an expression cassettefor a Myc polypeptide and/or a Sox polypeptide is not introduced intothe cells.

In some embodiments, the Klf polypeptide is Klf4 and the Oct polypeptideis Oct4.

In some embodiments, the non-pluripotent cells are somatic cells.

In some embodiments, the non-pluripotent cells are fibroblast cells.

In some embodiments, neither an expression cassette for expression of aMyc polypeptide nor an expression cassette for expression of a Klfpolypeptide are introduced into the non-pluripotent cells.

In some embodiments, the introducing step is performed in vivo. In someembodiments, the introducing step is performed in vitro.

In some embodiments, the method further comprises,

c) selecting cells that exhibit pluripotent stem cell characteristics.

In some embodiments, the non-pluripotent cells are obtained from ananimal; and the induced pluripotent stem cells are differentiated into adesired cell type.

In some embodiments, the desired cell type is introduced into theanimal. In some embodiments, the animal is a human. In some embodiments,the animal is a non-human animal.

In some embodiments, the selected cells do not comprise an exogenousexpression cassette for expression of Oct4.

In some embodiments, the agent inhibits H3K9 methylation. In someembodiments, the agent that inhibits H3K9 methylation is BIX01294.

In some embodiments, the cells are human cells. In some embodiments, thecells are mouse cells. In some embodiments, the non-pluripotent cellsare progenitor cells. In some embodiments, the progenitor cells areneural progenitor cells.

In some embodiments, the introducing step comprises introducing

a first vector comprising a promoter operably linked to a firstexpression cassette, the first expression cassette comprising apolynucleotide encoding Klf4;

a second vector comprising a promoter operably linked to a secondexpression cassette, the second expression cassette comprising apolynucleotide encoding Sox2; and

a third vector comprising a promoter operably linked to a thirdexpression cassette, the third expression cassette comprising apolynucleotide encoding c-Myc.

In some embodiments, the vectors are retroviral, lentiviral, adenoviralvectors, standard non-viral plasmid, or episomal expression vectors.

The present invention also comprises a mixture of mammalian cells and anagent that inhibits H3K9 methylation or promotes H3K9 demethylation,wherein the cells express at least one or more of an Oct polypeptide, aKlf polypeptide, a Sox polypeptide, and a Myc polypeptide; and/or are incontact with at least one or more of an exogenous Oct polypeptide, anexogenous Klf polypeptide, an exogenous Sox polypeptide, and anexogenous Myc polypeptide.

In some embodiments, the cells comprise a first, second and thirdrecombinant expression cassette, the first expression cassettecomprising a promoter operably linked to a polynucleotide encoding a Klfpolypeptide, the second expression cassette comprising a promoteroperably linked to a polynucleotide encoding a Sox polypeptide; and thethird expression cassette comprising a promoter operably linked to apolynucleotide encoding a Myc polypeptide.

In some embodiments, the agent inhibits H3K9 methylation. In someembodiments, the agent that inhibits H3K9 methylation is BIX01294.

In some embodiments, the cells comprise one or more retroviral,lentiviral, adenoviral, non-viral plasmid, or episomal expressionvector, the one or more retroviral, lentiviral, adenoviral, non-viralplasmid, or episomal expression vector comprising the first, second andthird expression cassette.

In some embodiments, the mixture comprises a first, second and thirdretroviral, lentiviral, adenoviral, non-viral plasmid, or episomalexpression vector, the first retroviral, lentiviral, adenoviral,non-viral plasmid, or episomal expression vector comprising the firstexpression cassette; the second retroviral, lentiviral, adenoviral,non-viral plasmid, or episomal expression vector comprising the secondexpression cassette; and the third retroviral, lentiviral, adenoviral,non-viral plasmid, or episomal expression vector comprising the thirdexpression cassette.

In some embodiments, the cells are human cells. In some embodiments, thecells are mouse cells. In some embodiments, the cells compriseprogenitor cells. In some embodiments, the progenitor cells are neuralprogenitor cells, skin progenitor cells or hair follicle progenitorcells.

In some embodiments, the Klf polypeptide is Klf 4, the Myc polypeptideis c-Myc, and the Sox polypeptide is Sox2.

The present invention also provides for a mammalian cell(s) thatendogenously expresses at least one of a protein selected from the groupconsisting of an Oct polypeptide, a Klf polypeptide, a Myc polypeptide,and a Sox polypeptide, wherein: the cell does not endogenously expressat least one protein of the group, wherein the protein not expressedendogenously is expressed from RNA encoded by a heterologous recombinantexpression cassette present in the cell, wherein the cell expressesendogenously or heterologously each of the Oct polypeptide, the Klfpolypeptide, the Myc polypeptide, and the Sox polypeptide; andexpression of the protein from the heterologous expression cassetteresults in reprogramming or dedifferentiation of the cell from anon-pluripotent cell to a pluripotent stem cell.

In some embodiments, the Oct polypeptide is Oct 4, the Klf polypeptideis Klf 4, the Myc polypeptide is c-Myc, and the Sox polypeptide is Sox2.In some embodiments, the cell endogenously expresses a Sox polypeptideand a Myc polypeptide and heterologously expresses an Oct polypeptideand a Klf polypeptide. In some embodiments, the Oct polypeptide is Oct4,the Klf polypeptide is Klf 4, the Myc polypeptide is c-Myc, and the Soxpolypeptide is Sox2.

The present invention also provides for methods of inducing Oct4expression in a cell. In some embodiments, the method comprising,contacting the cell with an agent that inhibits H3K9 methylation orpromotes H3K9 demethylation, thereby inducing Oct4 expression in thecell.

In some embodiments, the cell does not express Oct4 immediately prior tothe contacting step. In some embodiments, the cells that are contactedare not pluripotent cells. In some embodiments, the cells are induced topluripotency after the contacting step.

The present invention also provides methods for inducing non-pluripotentcells into pluripotent cells. In some embodiments, the method comprisescontacting the non-pluripotent cells with one or more agents that inducepluripotency and/or introducing expression cassettes into the cells toexpress proteins that induce pluripotency, wherein the cells are notcultured on feeder cells and the cells are attached to a solid culturesurface. In some embodiments, the method further comprises screening thecontacted cells for pluripotent stem cell characteristics.

In some embodiments, the cells are attached to the solid culture surfaceby a molecular tether, the molecular tether selected form the groupconsisting of matrigel, an extracellular matrix (ECM) or ECM analog,laminin, fibronectin, and collagen.

In some embodiments, the contacting step comprises the steps of

a) introducing one or more expression cassettes for expression of a Klfpolypeptide, an Oct polypeptide, a Myc polypeptide, and a Soxpolypeptide into the non-pluripotent cells;

b) contacting the cells with an agent that inhibits H3K9 methylation orpromotes H3K9 demethylation, thereby producing induced pluripotent stemcells.

The present invention also provides methods of producing inducedpluripotent stem cells from mammalian non-pluripotent cells. In someembodiments, the method comprises

a) contacting the cells with at least one (e.g., one, two, three, fouror more) of:

-   -   an agent that inhibits H3K9 methylation or promotes H3K9        demethylation (including but not limited to BIX);    -   an L-type Ca channel agonist (including but not limited to        BayK);    -   an activator of the cAMP pathway (including but not limited to        forskolin);    -   DNA methyltransferase (DNMT) inhibitor (including but not        limited to RG108 or 5-aza-C);    -   nuclear receptor ligand (including but not limited to        dexamethasone);    -   a GSK3 inhibitor (including but not limited to CHIR99021);    -   a MEK inhibitor,    -   a TGFβ receptor/ALK5 inhibitor (including but not limited to        SB431542, A-83-01, or        2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine),    -   a HDAC inhibitor (including but not limited to TSA, VPA, sodium        butyrate, SAHA etc); and    -   an Erk inhibitor.        thereby producing induced pluripotent stem cells.

In some embodiments, the method further comprises screening thecontacted cells for pluripotent stem cell characteristics.

In some embodiments, the method comprises

a) contacting the cells with:

-   -   i. an agent that inhibits H3K9 methylation or promotes H3K9        demethylation; and    -   ii. and agent selected from the group consisting of        -   an L-type Ca channel agonist (including but not limited to            BayK);        -   an activator of the cAMP pathway (including but not limited            to forskolin);        -   DNA methyltransferase (DNMT) inhibitor (including but not            limited to RG108 or 5-aza-C);        -   nuclear receptor ligand (including but not limited to            dexamethasone);        -   a GSK3 inhibitor (including but not limited to CHIR99021);        -   a MEK inhibitor,        -   a TGFβ receptor/ALK5 inhibitor (including but not limited to            SB431542, A-83-01, or            2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine),        -   a HDAC inhibitor (including but not limited to TSA, VPA,            sodium butyrate, SAHA etc); and        -   an Erk inhibitor.

In some embodiments, the method further comprises

b) introducing one or more expression cassettes for expression of a Klfpolypeptide, an Oct polypeptide, a Myc polypeptide, and/or a Soxpolypeptide into the non-pluripotent cells.

In some embodiments, Klf4 and Oct 4 are introduced into the cells (andoptionally, Myc and Sox are not).

The present invention also provides mixtures of mammalian cells and atleast one (e.g., one, two, three, four or more) of:

-   -   an agent that inhibits H3K9 methylation or promotes H3K9        demethylation,    -   an L-type Ca channel agonist;    -   an activator of the cAMP pathway;    -   DNA methyltransferase (DNMT) inhibitor;    -   nuclear receptor ligand;    -   a GSK3 inhibitor;    -   a MEK inhibitor,    -   a TGFβ receptor/ALK5 inhibitor,    -   a HDAC inhibitor; and    -   an Erk inhibitor.

In some embodiments, the mixture comprises

-   -   i. an agent that inhibits H3K9 methylation or promotes H3K9        demethylation, and    -   ii. an agent selected from the group consisting of        -   an L-type Ca channel agonist;        -   an activator of the cAMP pathway;        -   DNA methyltransferase (DNMT) inhibitor;        -   nuclear receptor ligand;        -   a GSK3 inhibitor;        -   a MEK inhibitor,        -   a TGFβ receptor/ALK5 inhibitor,        -   a HDAC inhibitor; and        -   an Erk inhibitor.

In some embodiments, the cells comprise a heterologous expressioncassette for expression of an Oct polypeptide and a heterologousexpression cassette for expression of a Sox polypeptide.

In some embodiments, the cells are non-pluripotent cells. In someembodiments, the cells are fibroblast cells.

In some embodiments, neither an expression cassette for expression of aMyc polypeptide nor an expression cassette for expression of a Klfpolypeptide are introduced into the non-pluripotent cells.

The present invention also provides compositions comprising an agentthat inhibits H3K9 methylation and an agent selected from the groupconsisting of

-   -   an L-type Ca channel agonist;    -   an activator of the cAMP pathway;    -   DNA methyltransferase (DNMT) inhibitor;    -   nuclear receptor ligand;    -   a GSK3 inhibitor;    -   a MEK inhibitor,    -   a TGFβ receptor/ALK5 inhibitor,    -   a HDAC inhibitor; and    -   an Erk inhibitor.

The present invention also provides kits comprising

i. an agent that inhibits H3K9 methylation or promotes H3K9demethylation; and

ii. an agent selected from the group consisting of

-   -   an L-type Ca channel agonist;    -   an activator of the cAMP pathway;    -   DNA methyltransferase (DNMT) inhibitor;    -   nuclear receptor ligand;    -   a GSK3 inhibitor;    -   a MEK inhibitor,    -   a TGFβ receptor/ALK5 inhibitor,    -   a HDAC inhibitor; and    -   an Erk inhibitor.

In some embodiments, the kits further comprise mammalian cells.

Some embodiments of the invention are set forth in claim format directlybelow:

1. A method of producing induced pluripotent stem cells from mammaliannon-pluripotent cells, the method comprising,

a) introducing one or more of an Oct polypeptide, a Klf polypeptide, aMyc polypeptide, and a Sox polypeptide into the non-pluripotent cells;

b) contacting the cells with an agent that inhibits H3K9 methylation orpromotes H3K9 demethylation, thereby producing induced pluripotent stemcells.

2. The method of claim 1, wherein step a) comprises contacting thenon-pluripotent cells with one or more exogenous polypeptides selectedfrom a Klf polypeptide, an Oct polypeptide, a Myc polypeptide, and a Soxpolypeptide.

3. The method of claim 2, wherein step a) comprises at least two cyclesof:

i. contacting the non-pluripotent cells with one or more exogenouspolypeptides selected from a Klf polypeptide, an Oct polypeptide, a Mycpolypeptide, and a Sox polypeptide;

ii. followed by culturing the cells in the absence of the exogenouspolypeptides.

4. The method of claim 1, wherein step a) comprises introducing one ormore expression cassettes for expression of a Klf polypeptide, an Octpolypeptide, a Myc polypeptide, and a Sox polypeptide into thenon-pluripotent cells.

5. The method of claim 1, wherein an expression cassette for expressionof an Oct polypeptide and an expression cassette for expression of a Soxpolypeptide are introduced into the non-pluripotent cells.

6. The method of claim 1, wherein the introducing step comprisesintroducing into the non-pluripotent cells one or more expressioncassettes for expression of a KLF polypeptide and an Oct polypeptide,wherein an expression cassette for a Myc polypeptide and/or a Soxpolypeptide is not introduced into the cells.7. The method of any of claims 1-6, wherein the Klf polypeptide is Klf4and the Oct polypeptide is Oct4.8. The method of any of claims 1-7, wherein the non-pluripotent cellsare somatic cells.9. The method of any of claims 1-7, wherein the non-pluripotent cellsare fibroblast cells.10. The method of any of claims 1-7, wherein neither an expressioncassette for expression of a Myc polypeptide nor an expression cassettefor expression of a Klf polypeptide are introduced into thenon-pluripotent cells.11. The method of any of claims 1-10, wherein the introducing step isperformed in vivo.12. The method of any of claims 1-10, wherein the introducing step isperformed in vitro.13. The method of claim any of claims 1-12, further comprising,c) selecting cells that exhibit pluripotent stem cell characteristics.14. The method of any of claims 1-13, wherein the non-pluripotent cellsare obtained from an animal; andthe induced pluripotent stem cells are differentiated into a desiredcell type.15. The method of claim 14, wherein the desired cell type is introducedinto the animal.16. The method of claim 15, wherein the animal is a human.17. The method of claim 15, wherein the animal is a non-human animal.18. The method of claim 13, wherein the selected cells do not comprisean exogenous expression cassette for expression of Oct4.19. The method of any of claims 1-8, wherein the agent inhibits H3K9methylation.20. The method of claim 19, wherein the agent that inhibits H3K9methylation is BIX01294.21. The method of any of claims 1-20, wherein the cells are human cells.22. The method of any of claims 1-20, wherein the cells are mouse dog,cow, pig, rat and non-human primate cells.23. The method of claim any of claims 1-22, wherein the non-pluripotentcells are progenitor cells.24. The method of claim 23, wherein the progenitor cells are neuralprogenitor cells, skin progenitor cells or hair follicle progenitorcells.25. The method of any of claim 1 or 4-24, wherein the introducing stepcomprises introducinga first vector comprising a promoter operably linked to a firstexpression cassette, the first expression cassette comprising apolynucleotide encoding Klf4;a second vector comprising a promoter operably linked to a secondexpression cassette, the second expression cassette comprising apolynucleotide encoding Sox2; anda third vector comprising a promoter operably linked to a thirdexpression cassette, the third expression cassette comprising apolynucleotide encoding c-Myc.26. The method of claim any of claim 1 or 4-25, wherein the vectors areretroviral, lentiviral, adenoviral, non-viral plasmid, or episomalexpression vectors.27. A method for screening for agents that induce reprogramming ordedifferentiation of mammalian cells into pluripotent stem cells, themethod comprising,a) introducing at least one of, but not all of, an Oct polypeptide, aKlf polypeptide, a Myc polypeptide, and a Sox polypeptide intonon-pluripotent cells to generate transfected cells;b) contacting the transfected cells to a library of different agents;c) screening the contacted cells for pluripotent stem cellcharacteristics; andd) correlating the development of stem cell characteristics with aparticular agent from the library, thereby identifying an agent thatstimulates dedifferentiation of cells into pluripotent stem cells.28. The method of claim 27, wherein step a) comprises introducing one ormore expression cassettes for expression of the at least one of, but notall of, an Oct polypeptide, a Klf polypeptide, a Myc polypeptide, and aSox polypeptide into the non-pluripotent cells.29. The method of claim 27, wherein step a) comprises introducing atleast one of, but not all of, an exogenous Oct polypeptide, an exogenousKlf polypeptide, an exogenous Myc polypeptide, and an exogenous Soxpolypeptide into the non-pluripotent cells.30. The method of any of claims 27-29, wherein the particular agent isbetween 50-1500 daltons.31. The method of claim 27, wherein step a) comprises introducing twoexpression cassettes into the cells, wherein each expression cassettecomprises a polynucleotide encoding a different protein, wherein theprotein is selected from the group consisting of an Oct polypeptide, aKlf polypeptide, a Myc polypeptide, and a Sox polypeptide, and theremaining members of the group are not introduced into the cells.32. The method of claim 27, wherein step a) comprises introducing threeexpression cassettes into the cells, wherein each expression cassettecomprises a polynucleotide encoding a different protein, wherein theprotein is selected from the group consisting of an Oct polypeptide, aKlf polypeptide, a Myc polypeptide, and a Sox polypeptide, and theremaining member of the group is not introduced into the cells.33. The method of any of claims 27-32, wherein the cells are humancells.34. The method of any of claims 27-33, wherein the cells are non-humanmammalian cells.35. The method of any of claims 27-34, wherein the non-pluripotent cellsare progenitor cells.36. The method of claim 35, wherein the progenitor cells are neuralprogenitor cells, skin progenitor cells or hair follicle progenitorcells.37. The method of any of claims 27-36, wherein the Oct polypeptide isOct4, the Klf polypeptide is Klf 4, the Myc polypeptide is c-Myc, andthe Sox polypeptide is Sox2.38. A method of screening for mammalian cells with pluripotent stem cellcharacteristics, the method comprising,a) contacting cells with a MAPK/ERK kinase (MEK) inhibitor such thatgrowth of non-pluripotent cells is inhibited and growth of pluripotentstem cells is promoted; andb) screening the contacted cells for pluripotent stem cellcharacteristics.39. The method of claim 38, comprising,contacting the cells with a library of agents prior to step a);and, following step b), selecting an agent that induces pluripotent stemcell based on the results of step b).40. The method of any of claims 38-39, wherein the cells are humancells.41. The method of any of claims 38-39, wherein the cells are mouse, dog,cow, pig, rat and non-human primate cells.42. The method of any of claims 38-41, wherein the MEK inhibitor isPD0325901.43. A mixture of mammalian cells and an agent that inhibits H3K9methylation or promotes H3K9 demethylation, wherein the cells:express at least one or more of an Oct polypeptide, a Klf polypeptide, aSox polypeptide, and a Myc polypeptide; and/orare in contact with at least one or more of an exogenous Octpolypeptide, an exogenous Klf polypeptide, an exogenous Sox polypeptide,and an exogenous Myc polypeptide.44. The mixture of claim 43, wherein the cells comprise a first, secondand third recombinant expression cassette,the first expression cassette comprising a promoter operably linked to apolynucleotide encoding a Klf polypeptide,the second expression cassette comprising a promoter operably linked toa polynucleotide encoding a Sox polypeptide; andthe third expression cassette comprising a promoter operably linked to apolynucleotide encoding a Myc polypeptide.45. The mixture of any of claims 43-44, wherein the agent inhibits H3K9methylation.46. The method of claim 45, wherein the agent that inhibits H3K9methylation is BIX01294.47. The mixture of any of claims 43-46, wherein the cells comprise oneor more retroviral, lentiviral, adenoviral, non-viral plasmid, orepisomal expression vector, the one or more retroviral, lentiviral,adenoviral, non-viral plasmid, or episomal expression vector comprisingthe first, second and third expression cassette.48. The mixture of claim 47, comprising a first, second and thirdretroviral, lentiviral, adenoviral, non-viral plasmid, or episomalexpression vector,the first retroviral, lentiviral, adenoviral, non-viral plasmid, orepisomal expression vector comprising the first expression cassette;the second retroviral, lentiviral, adenoviral, non-viral plasmid, orepisomal expression vector comprising the second expression cassette;andthe third retroviral, lentiviral, adenoviral, non-viral plasmid, orepisomal expression vector comprising the third expression cassette.49. The mixture of claim any of claims 43-48, wherein the cells arehuman cells.50. The mixture of claim any of claims 43-48, wherein the cells aremouse dog, cow, pig, rat and non-human primate cells.51. The mixture of any of claims 43-50, wherein the cells compriseprogenitor cells.52. The mixture of claim 51, wherein the progenitor cells are neuralprogenitor cells, skin progenitor cells or hair follicle progenitorcells.53. The mixture of claim any of claims 43-52, wherein the Klfpolypeptide is Klf 4, the Myc polypeptide is c-Myc, and the Soxpolypeptide is Sox2.54. A mammalian cell that endogenously expresses at least one of aprotein selected from the group consisting of an Oct polypeptide, a Klfpolypeptide, a Myc polypeptide, and a Sox polypeptide,wherein:the cell does not endogenously express at least one protein of thegroup, wherein the protein not expressed endogenously is expressed fromRNA encoded by a heterologous recombinant expression cassette present inthe cell,the cell expresses endogenously or heterologously each of the Octpolypeptide, the Klf polypeptide, the Myc polypeptide, and the Soxpolypeptide; andexpression of the protein from the heterologous expression cassetteresults in reprogramming or dedifferentiation of the cell from anon-pluripotent cell to a pluripotent stem cell.55. The cell of claim 54, wherein the Oct polypeptide is Oct 4, the Klfpolypeptide is Klf 4, the Myc polypeptide is c-Myc, and the Soxpolypeptide is Sox2.56. The cell of any of claims 54-55, wherein the cell endogenouslyexpresses a Sox polypeptide and a Myc polypeptide and heterologouslyexpresses an Oct polypeptide and a Klf polypeptide.57. The cell of claim 56, wherein the Oct polypeptide is Oct4, the Klfpolypeptide is Klf 4, the Myc polypeptide is c-Myc, and the Soxpolypeptide is Sox2.58. A method of inducing Oct4 expression in a cell, the methodcomprising, contacting the cell with an agent that inhibits H3K9methylation or promotes H3K9 demethylation, thereby inducing Oct4expression in the cell.59. The method of claim 58, wherein the cell does not express Oct4immediately prior to the contacting step.60. The method of any of claims 58-59, wherein the cells that arecontacted are not pluripotent cells.61. The method of any of claims 58-59, wherein the cells are induced topluripotency after the contacting step.62. A method for inducing non-pluripotent cells into pluripotent cells,the method comprising,contacting the non-pluripotent cells with one or more agents that inducepluripotency and/or introducing expression cassettes into the cells toexpress proteins that induce pluripotency, wherein the cells are notcultured on feeder cells and the cells are attached to a solid culturesurface.63. The method of claim 62, wherein the cells are attached to the solidculture surface by a molecular tether, the molecular tether selectedform the group consisting of matrigel, an extracellular matrix (ECM) orECM analog, laminin, fibronectin, and collagen.64. The method of claim 62, wherein the contacting step comprises thesteps of claim 1, or claims dependent on claim 1.65. A method of producing induced pluripotent stem cells from mammaliannon-pluripotent cells, the method comprising,a) contacting the cells with at least two of:

-   -   an agent that inhibits H3K9 methylation or promotes H3K9        demethylation;    -   an L-type Ca channel agonist;    -   an activator of the cAMP pathway;    -   DNA methyltransferase (DNMT) inhibitor;    -   a nuclear receptor ligand;    -   a GSK3 inhibitor;    -   a MEK inhibitor;    -   a TGFβ receptor/ALK5 inhibitor;    -   a HDAC inhibitor; and    -   an erk inhibitor,        thereby producing induced pluripotent stem cells.        66. The method of claim 65, wherein the contacting step        comprises        a) contacting the cells with at least two of:    -   i. an agent that inhibits H3K9 methylation or promotes H3K9        demethylation; and    -   ii. and agent selected from the group consisting of        -   an L-type Ca channel agonist;        -   an activator of the cAMP pathway;        -   DNA methyltransferase (DNMT) inhibitor;        -   a nuclear receptor ligand;        -   a GSK3 inhibitor;        -   a MEK inhibitor;        -   a TGFβ receptor/ALK5 inhibitor;        -   a HDAC inhibitor; and        -   an erk inhibitor.            67. The method of claim 65 or 66, further comprising            b) introducing one or more expression cassettes for            expression of a Klf polypeptide, an Oct polypeptide, a Myc            polypeptide, and/or a Sox polypeptide into the            non-pluripotent cells.            68. The method of any of claims 65-67, wherein Klf4 and Oct            4 are introduced into the cells.            69. A mixture of mammalian cells and at least two of    -   an agent that inhibits H3K9 methylation or promotes H3K9        demethylation,    -   an L-type Ca channel agonist;    -   an activator of the cAMP pathway;    -   DNA methyltransferase (DNMT) inhibitor;    -   a nuclear receptor ligand;    -   a GSK3 inhibitor;    -   a MEK inhibitor;    -   a TGFβ receptor/ALK5 inhibitor;    -   a HDAC inhibitor; and    -   an erk inhibitor.        70. The mixture of claim 69, wherein the mixture comprises:    -   i. an agent that inhibits H3K9 methylation or promotes H3K9        demethylation, and    -   ii. an agent selected from the group consisting of        -   an L-type Ca channel agonist;        -   an activator of the cAMP pathway;        -   DNA methyltransferase (DNMT) inhibitor;        -   a nuclear receptor ligand;        -   a GSK3 inhibitor;        -   a MEK inhibitor;        -   a TGFβ receptor/ALK5 inhibitor;        -   a HDAC inhibitor; and        -   an erk inhibitor.            71. The mixture of any of claims 69-70, wherein the cells            comprise a heterologous expression cassette for expression            of an Oct polypeptide and a heterologous expression cassette            for expression of a Sox polypeptide            72. The mixture of any of claims 69-71, wherein the cells            are non-pluripotent cells.            73. The mixture of any of claims 69-72, wherein the cells            are fibroblast cells.            74. The mixture of any of claims 69-73, wherein neither an            expression cassette for expression of a Myc polypeptide nor            an expression cassette for expression of a Klf polypeptide            are introduced into the non-pluripotent cells.            75. A composition comprising at least two of:    -   an agent that inhibits H3K9 methylation;    -   an L-type Ca channel agonist;    -   an activator of the cAMP pathway;    -   DNA methyltransferase (DNMT) inhibitor;    -   a nuclear receptor ligand;    -   a GSK3 inhibitor;    -   a MEK inhibitor;    -   a TGFβ receptor/ALK5 inhibitor;    -   a HDAC inhibitor; and    -   an erk inhibitor.        76. The composition of claim 75, comprising    -   i. an agent that inhibits H3K9 methylation and    -   ii. an agent selected from the group consisting of        -   an L-type Ca channel agonist;        -   an activator of the cAMP pathway;        -   DNA methyltransferase (DNMT) inhibitor;        -   a nuclear receptor ligand;        -   a GSK3 inhibitor;        -   a MEK inhibitor;        -   a TGFβ receptor/ALK5 inhibitor;        -   a HDAC inhibitor; and        -   an erk inhibitor.            77. A kit comprising at least two of:    -   an agent that inhibits H3K9 methylation;    -   an L-type Ca channel agonist;    -   an activator of the cAMP pathway;    -   DNA methyltransferase (DNMT) inhibitor;    -   a nuclear receptor ligand;    -   a GSK3 inhibitor;    -   a MEK inhibitor;    -   a TGFβ receptor/ALK5 inhibitor;    -   a HDAC inhibitor; and    -   an erk inhibitor.        78. The kit of claim 77, comprising    -   i. an agent that inhibits H3K9 methylation and    -   ii. an agent selected from the group consisting of        -   an L-type Ca channel agonist;        -   an activator of the cAMP pathway;        -   DNA methyltransferase (DNMT) inhibitor;        -   a nuclear receptor ligand;        -   a GSK3 inhibitor;        -   a MEK inhibitor;        -   a TGFβ receptor/ALK5 inhibitor;        -   a HDAC inhibitor; and        -   an erk inhibitor.            79. The kit of claim 77 or 78, further comprising mammalian            cells.

Other embodiments of the invention will be clear from a reading of theentire application.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 Generation of iPS cells from defined primary neural progenitorcells by Oct4/Klf4 viral transduction and BIX01294 treatment. Acomparison of the number of GFP+ iPS cell colonies generated from3.5×10⁴ primary OG2 neural progenitor cells by retroviral transductionof Oct4/Klf4/Sox2/c-Myc, Oct4/Klf4/Sox2, or Oct4/Klf4 with or withoutBIX01294 treatment.

FIG. 2 Generation of iPS cells from primary neural progenitor cells byKlf4/Sox2/c-Myc viral transduction and BIX01294 treatment. The number ofGFP+ iPS cell colonies generated from 3.5×10⁴ primary OG2 neuralprogenitor cells by retroviral transduction of Klf4/Sox2/c-Myc with orwithout BIX01294 treatment.

FIG. 3. Generation of OK2B iPSCs from primary OG2 MEFs. Bar graphshowing the average number of GFP⁺ colony induced from OG2-MEFs in 3independent experiments. This graph shows data for OG2 MEF cellstransduced with 4 factors (Oct4, Klf4, Sox2, and cMyc; 4F); transducedwith OK (OK); transduced with OK and treated with 1 μM BIX (OK+BIX);transduced with OK and treated with 1 μM BIX+2 μM BayK (OK+BIX+BayK);transduced with OK and treated with 1 μM BIX+0.04 μM RG108(OK+BIX+RG108), n=3, error bar represents standard deviation ascalculated with Excel.

FIG. 4. OK2B iPSCs have a transcriptional profile similar to the one ofmESCs. (A) RT-PCR analysis of OK2B iPSCs indicated they express genesspecific to pluripotent mESCs. R1 mESCs were used as positive controlwhile OG2 MEFs were used as negative control. GAPDH was used as loadingcontrol. (B) Bisulfite sequencing revealed that OK2B iPSCs' nanogpromoter is demethylated, further suggesting a reactivation ofendogenous genes specific to mESCs. Schematic depiction of the cytosinepresent in the region of the Nanog promoter that was amplified for thisanalysis. Open circle indicates demethylated cytosine, whilefilled/black circle indicates methylated cytosine.

FIG. 5. Treatment with BIX, BayK or a combination of both does notincrease the proliferation of mES cells. Scatter graph showing R1 mEScell number after treatment with DMSO (control), 2 μM BayK, 1 BayK, 1 μMBIX and a combination of both (BayK+BIX). n=3. The error bars representstandard deviation as calculated in Excel. No significant difference wasobtained for each treatment compared to DMSO as calculated using t-testin Excel.

FIG. 6. RT-PCR analysis for Sox2 expression after compound treatment.OG2^(+/−)/ROSA26^(+/−) (OG2) MEFs were treated with DMSO (control), 1 μMBIX, 2 μM BayK, and a combination of both for 6 days. RNA was thenextracted using Qiagen RNAeasy Mini Kit. Sox2 expression was assessedthrough semi-quantitative PCR. OK2B iPSCs p37 and R1 were used aspositive control, GAPDH as loading control.

DEFINITIONS

An “Oct polypeptide” refers to any of the naturally-occurring members ofOctomer family of transcription factors, or variants thereof thatmaintain transcription factor activity, similar (within at least 50%,80%, or 90% activity) compared to the closest related naturallyoccurring family member, or polypeptides comprising at least theDNA-binding domain of the naturally occurring family member, andoptionally comprising a transcriptional activation domain. Exemplary Octpolypeptides include, e.g., Oct3/4 (referred to herein as “Oct4”), whichcontains the POU domain. See, Ryan, A. K. & Rosenfeld, M. G. Genes Dev.11, 1207-1225 (1997). In some embodiments, variants have at least 90%amino acid sequence identity across their whole sequence compared to anaturally occurring Oct polypeptide family member such as to thoselisted above.

A “Klf polypeptide” refers to any of the naturally-occurring members ofthe family of Krüppel-like factors (Klfs), zinc-finger proteins thatcontain amino acid sequences similar to those of the Drosophilaembryonic pattern regulator Krüppel, or variants of thenaturally-occurring members that maintain transcription factor activitysimilar (within at least 50%, 80%, or 90% activity) compared to theclosest related naturally occurring family member, or polypeptidescomprising at least the DNA-binding domain of the naturally occurringfamily member, and optionally comprising a transcriptional activationdomain. See, Dang, D. T., Pevsner, J. & Yang, V. W. Cell Biol. 32,1103-1121 (2000). Exemplary Klf family members include, e.g., Klf1,Klf4, and Klf5, each of which have been shown to be able to replace eachother to result in iPS cells. See, Nakagawa, et al., NatureBiotechnology 26:101-106 (2007). In some embodiments, variants have atleast 90% amino acid sequence identity across their whole sequencecompared to a naturally occurring Klf polypeptide family member such asto those listed above. To the extent a KLF polypeptide is describedherein, it can be replaced with an Essrb. Thus, it is intended that foreach Klf polypeptide embodiment described herein is equally describedfor use of Essrb in the place of a Klf4 polypeptide.

A “Myc polypeptide” refers any of the naturally-occurring members of theMyc family (see, e.g., Adhikary, S. & Eilers, M. Nat. Rev. Mol. Cell.Biol. 6:635-645 (2005)), or variants thereof that maintain transcriptionfactor activity similar (within at least 50%, 80%, or 90% activity)compared to the closest related naturally occurring family member, orpolypeptides comprising at least the DNA-binding domain of the naturallyoccurring family member, and optionally comprising a transcriptionalactivation domain. Exemplary Myc polypeptides include, e.g., c-Myc,N-Myc and L-Myc. In some embodiments, variants have at least 90% aminoacid sequence identity across their whole sequence compared to anaturally occurring Myc polypeptide family member such as to thoselisted above.

A “Sox polypeptide” refers to any of the naturally-occurring members ofthe SRY-related HMG-box (Sox) transcription factors, characterized bythe presence of the high-mobility group (HMG) domain, or variantsthereof that maintain transcription factor activity similar (within atleast 50%, 80%, or 90% activity) compared to the closest relatednaturally occurring family member, or polypeptides comprising at leastthe DNA-binding domain of the naturally occurring family member, andoptionally comprising a transcriptional activation domain. See, e.g.,Dang, D. T., et al., Int. J. Biochem. Cell Biol. 32:1103-1121 (2000).Exemplary Sox polypeptides Include, e.g., Sox1, Sox2, Sox3, Sox15, orSox18, each of which have been shown to be able to replace each other toresult in iPS cells. See, Nakagawa, et al., Nature Biotechnology26:101-106 (2007). In some embodiments, variants have at least 90% aminoacid sequence identity across their whole sequence compared to anaturally occurring Sox polypeptide family member such as to thoselisted above.

“H3K9” refers to histone H3 lysine 9. H3K9 can be di-methylated at K9.See, e.g., Kubicek, et al., Mol. Cell 473-481 (2007).

The term “pluripotent” or “pluripotency” refers to cells with theability to give rise to progeny that can undergo differentiation, underthe appropriate conditions, into cell types that collectivelydemonstrate characteristics associated with cell lineages from all ofthe three germinal layers (endoderm, mesoderm, and ectoderm).Pluripotent stem cells can contribute to many or all tissues of aprenatal, postnatal or adult animal. A standard art-accepted test, suchas the ability to form a teratoma in 8-12 week old SCID mice, can beused to establish the pluripotency of a cell population, howeveridentification of various pluripotent stem cell characteristics can alsobe used to detect pluripotent cells.

“Pluripotent stem cell characteristics” refer to characteristics of acell that distinguish pluripotent stem cells from other cells. Theability to give rise to progeny that can undergo differentiation, underthe appropriate conditions, into cell types that collectivelydemonstrate characteristics associated with cell lineages from all ofthe three germinal layers (endoderm, mesoderm, and ectoderm) is apluripotent stem cell characteristic. Expression or non-expression ofcertain combinations of molecular markers are also pluripotent stem cellcharacteristics. For example, human pluripotent stem cells express atleast some, and optionally all, of the markers from the followingnon-limiting list: SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49/6E, ALP,Sox2, E-cadherin, UTF-1, Oct4, Rex1, and Nanog. Cell morphologiesassociated with pluripotent stem cells are also pluripotent stem cellcharacteristics.

The term “library” is used according to its common usage in the art, todenote a collection of molecules, optionally organized and/or catalogedin such a way that individual members can be identified. Libraries caninclude, but are not limited to, combinatorial chemical libraries,natural products libraries, and peptide libraries.

A “recombinant” polynucleotide is a polynucleotide that is not in itsnative state, e.g., the polynucleotide comprises a nucleotide sequencenot found in nature, or the polynucleotide is in a context other thanthat in which it is naturally found, e.g., separated from nucleotidesequences with which it typically is in proximity in nature, or adjacent(or contiguous with) nucleotide sequences with which it typically is notin proximity. For example, the sequence at issue can be cloned into avector, or otherwise recombined with one or more additional nucleicacid.

“Expression cassette” refers to a polynucleotide comprising a promoteror other regulatory sequence operably linked to a sequence encoding aprotein.

The terms “promoter” and “expression control sequence” are used hereinto refer to an array of nucleic acid control sequences that directtranscription of a nucleic acid. As used herein, a promoter includesnecessary nucleic acid sequences near the start site of transcription,such as, in the case of a polymerase II type promoter, a TATA element. Apromoter also optionally includes distal enhancer or repressor elements,which can be located as much as several thousand base pairs from thestart site of transcription. Promoters include constitutive andinducible promoters. A “constitutive” promoter is a promoter that isactive under most environmental and developmental conditions. An“inducible” promoter is a promoter that is active under environmental ordevelopmental regulation. The term “operably linked” refers to afunctional linkage between a nucleic acid expression control sequence(such as a promoter, or array of transcription factor binding sites) anda second nucleic acid sequence, wherein the expression control sequencedirects transcription of the nucleic acid corresponding to the secondsequence.

A “heterologous sequence” or a “heterologous nucleic acid”, as usedherein, is one that originates from a source foreign to the particularhost cell, or, if from the same source, is modified from its originalform. Thus, a heterologous expression cassette in a cell is anexpression cassette that is not endogenous to the particular host cell,for example by being linked to nucleotide sequences from an expressionvector rather than chromosomal DNA, being linked to a heterologouspromoter, being linked to a reporter gene, etc.

The terms “agent” or “test compound” refer to any compound useful in thescreening assays described herein. An agent can be, for example, anorganic compound (e.g., a small molecule such as a drug), a polypeptide(e.g., a peptide or an antibody), a nucleic acid (e.g., DNA, RNA,double-stranded, single-stranded, an oligonucleotide, antisense RNA,small inhibitory RNA, micro RNA, a ribozyme, etc.), an oligosaccharide,a lipid. Usually, the agents used in the present screening methods havea molecular weight of less than 10,000 daltons, for example, less than8000, 6000, 4000, 2000 daltons, e.g., between 50-1500, 500-1500,200-2000, 500-5000 daltons. The test compound can be in the form of alibrary of test compounds, such as a combinatorial or randomized librarythat provides a sufficient range of diversity. Test compounds areoptionally linked to a fusion partner, e.g., targeting compounds, rescuecompounds, dimerization compounds, stabilizing compounds, addressablecompounds, and other functional moieties. Conventionally, new chemicalentities with useful properties are generated by identifying a testcompound (called a “lead compound”) with some desirable property oractivity, e.g., ability to induce pluripotency under certain conditionssuch as are described herein, creating variants of the lead compound,and evaluating the property and activity of those variant compounds.Often, high throughput screening (HTS) methods are employed for such ananalysis.

The terms “nucleic acid” and “polynucleotide” are used interchangeablyherein to refer to deoxyribonucleotides or ribonucleotides and polymersthereof in either single- or double-stranded form. The term encompassesnucleic acids containing known nucleotide analogs or modified backboneresidues or linkages, which are synthetic, naturally occurring, andnon-naturally occurring, which have similar binding properties as thereference nucleic acid, and which are metabolized in a manner similar tothe reference nucleotides. Examples of such analogs include, withoutlimitation, phosphorothioates, phosphoramidates, methyl phosphonates,chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleicacids (PNAs).

Unless otherwise indicated, a particular nucleic acid sequence alsoencompasses conservatively modified variants thereof (e.g., degeneratecodon substitutions) and complementary sequences, as well as thesequence explicitly indicated. Specifically, degenerate codonsubstitutions may be achieved by generating sequences in which the thirdposition of one or more selected (or all) codons is substituted withmixed-base and/or deoxyinosine residues (Batzer et al., Nucleic AcidRes. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608(1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)).

“Inhibitors,” “activators,” and “modulators” of expression or ofactivity are used to refer to inhibitory, activating, or modulatingmolecules, respectively, identified using in vitro and in vivo assaysfor expression or activity of a described target protein (or encodingpolynucleotide), e.g., ligands, agonists, antagonists, and theirhomologs and mimetics. The term “modulator” includes inhibitors andactivators. Inhibitors are agents that, e.g., inhibit expression or bindto, partially or totally block stimulation or protease inhibitoractivity, decrease, prevent, delay activation, inactivate, desensitize,or down regulate the activity of the described target protein, e.g.,antagonists. Activators are agents that, e.g., induce or activate theexpression of a described target protein or bind to, stimulate,increase, open, activate, facilitate, enhance activation or proteaseinhibitor activity, sensitize or up regulate the activity of describedtarget protein (or encoding polynucleotide), e.g., agonists. Modulatorsinclude naturally occurring and synthetic ligands, antagonists andagonists (e.g., small chemical molecules, antibodies and the like thatfunction as either agonists or antagonists). Such assays for inhibitorsand activators include, e.g., applying putative modulator compounds tocells expressing the described target protein and then determining thefunctional effects on the described target protein activity, asdescribed above. Samples or assays comprising described target proteinthat are treated with a potential activator, inhibitor, or modulator arecompared to control samples without the inhibitor, activator, ormodulator to examine the extent of effect. Control samples (untreatedwith modulators) are assigned a relative activity value of 100%.Inhibition of a described target protein is achieved when the activityvalue relative to the control is about 80%, optionally 50% or 25, 10%,5% or 1%. Activation of the described target protein is achieved whenthe activity value relative to the control is 110%, optionally 150%,optionally 200, 300%, 400%, 500%, or 1000-3000% or more higher.

DETAILED DESCRIPTION OF THE INVENTION

I. Introduction

The present invention is based, in part, on the surprising discoverythat small molecules may be used to mimic the effects of thetranscription factors involved in triggering induced pluripotent stemcells (iPS). For example, as described in detail herein, Oct4 can be“replaced” with a small molecule that reduced methylation of the histone3 lysine 9 (H3K9). Thus, for example, BIX01294, a small molecule thatspecifically inhibits G9a (a histone methyltransferase for H3K9), whencontacted to a mammalian cell in which Klf4, c-Myc and Sox2 has beenexpressed, results in induction of pluripotent stem cells.

These results are not only interesting with regard to the role ofmethylation of H3K9 and its involvement in cell programming, but alsoshows small molecules can be identified that replace transcriptionfactors previously shown to be essential for induction of pluripotentstem cells. This can be of particular interest where the goal isintroduction (or re-introduction) of induced pluripotent stem cells, orsubsequently differentiated cells, such a progenitor cells derived fromthe iPS cells, into a patient. As several (e.g., Oct4, Myc) of the fouriPS transcription factors have known oncogenic activities, it may bebeneficial to replace these factors with other less or non-oncogenicmolecules. Further, replacement of some or each of the four factors withsmall molecules that do not require transformation (and thus thepotential oncogenic effects of DNA insertion into the chromosome) willfurther help to reduce the possible cancer side effects of therapiesinvolving iPS.

The present invention also provides induced pluripotent cells in whichat least some of the iPS transcription factors are expressed atendogenous or lower levels and nevertheless are pluripotent. Thisinvention is based, in part, on the discovery that certain cells thatendogenously express Sox2 can be induced to pluripotency by introductionand heterologous expression of only Oct4 and Klf4.

Further, as shown herein, non-pluripotent cells that do not endogenouslyor heterologously express a Sox polypeptide (e.g., Sox2) can be inducedto pluripotency using the methods of the invention. For example,pluripotency can be induced by introduction of only Oct4 and Klf4 intonon-pluripotent cells (e.g., fibroblast cells) that are also contactedwith an agent that inhibit H3K9 methylation, and optionally alsocontacted with at least one of an L-type calcium channel agonist, anactivator of the cAMP pathway, a DNA methyltransferase (DNMT) inhibitor,a nuclear receptor agonist, a GSK3 inhibitor, or a MEK inhibitor. Theinventors have also found that combinations of agents such as a GSKinhibitor and a HDAC inhibitor; or a GSK inhibitor and a cAMP pathwayactivator; or a GSK inhibitor and an ALK5 inhibitor (with and without aG9a inhibitor) are effective in inducing pluripotency in cellsheterologously expressing Oct4 alone, or Oct4/Sox2 or Sox2/Klf4.Accordingly, the invention provides for mixtures of cells and agents,wherein the cells are initially not pluripotent cells (e.g., arenon-stem cells) and optionally heterologously or endogenously express orare otherwise in intact with Oct4 and/or Klf4, and wherein the agent(s)is one or more of an agent that inhibit H3K9 methylation, an L-type Cachannel agonist; an activator of the cAMP pathway; a DNAmethyltransferase (DNMT) inhibitor; a nuclear receptor ligand; a GSK3inhibitor; a MEK inhibitor, a TGFβ receptor/ALK5 inhibitor, a HDACinhibitor; and/or an Erk inhibitor.

As discussed further below, the invention also provides novel methods ofscreening for cells with pluripotent stem cell characteristics byculturing cells to be screened with a MEK inhibitor, an agent thatinhibits H3K9 methylation, an L-type Ca channel agonist; an activator ofthe cAMP pathway; a DNA methyltransferase (DNMT) inhibitor; a nuclearreceptor ligand; a GSK3 inhibitor; a MEK inhibitor, a TGFβ receptor/ALK5inhibitor, a HDAC inhibitor; or an Erk inhibitor. MEK inhibitors, forexample, inhibit growth of non-iPS cells while simultaneously promotinggrowth and stable reprogramming of iPS cells, thereby enriching aparticular cell mixture for cells with pluripotent stem cellcharacteristics.

II. Induction of Pluripotent Stem Cells

A. Heterologous/Endogenous Expression

In some embodiments of the invention, non-pluripotent cells areidentified that endogenously express at least one (and optionally, twoor three of) proteins from the group consisting of a Oct polypeptide, aKlf polypeptide, a Myc polypeptide, and a Sox polypeptide. The remaining(non-endogenously expressed) proteins from the group can then beheterologously expressed in the cells, and screened for re-programmingand/or dedifferentiation into pluripotent cells, optionally in thepresence of one or more of a MEK inhibitor, an agent that inhibits H3K9methylation, an L-type Ca channel agonist; an activator of the cAMPpathway; a DNA methyltransferase (DNMT) inhibitor; a nuclear receptorligand; a GSK3 inhibitor; a MEK inhibitor, a TGFβ receptor/ALK5inhibitor, a HDAC inhibitor; and/or an Erk inhibitor.

It is believed that any type of mammalian non-pluripotent cell can bescreened for protein expression and subsequently be converted to apluripotent cell. In some embodiments, the starting cells are isolatedprogenitor cells. Exemplary progenitor cells include, but are notlimited to, endoderm progenitor cells, mesoderm progenitor cells (e.g.,muscle progenitor cells, bone progenitor cells, blood progenitor cells),and ectoderm progenitor cells (e.g., epidermal tissue progenitor cellsand neural progenitor cells). Cells useful for these aspects of theinvention can be readily identified by screening cell lines forexpression of a Oct polypeptide, a Klf polypeptide, a Myc polypeptide,and a Sox polypeptide or by identifying cells with reduced promotermethylation (e.g., by DNA bisulfate sequencing) or identifying cellswith a modified histone state to determine the reduced silencing stateof these genes. Those transcription factors that are not expressedendogenously can then be expressed heterologously to induce pluripotencywithout heterologous expression of those factors already expressedendogenously.

As shown in the Examples, some cells (e.g., neural progenitor cells andfibroblasts (data not shown)) can be induced into pluripotency byheterologous expression of Oct4 and Klf4 only. This demonstrates thatSox and Myc proteins, and likely Oct and Klf proteins, need not all beoverexpressed (e.g., using a high expression viral vector) to achievepluripotency. Instead, some of these proteins can be expressed atendogenous or even lower detectable levels and still be eligible forconversion to pluripotent cells by heterologous expression of othermembers of the group. Thus, in some embodiments of the invention, cellsare identified that endogenously express a Sox polypeptide and/or a Mycpolypeptide, and an Oct polypeptide and a Klf polypeptide isheterologously expressed in the cell, thereby inducing conversion of thecell into a pluripotent cell. In some embodiments, cells are identifiedthat endogenously express an Oct polypeptide and/or a Klf polypeptideand/or a Myc polypeptide, and a Sox polypeptide is heterologouslyexpressed in the cell, thereby inducing conversion of the cell into apluripotent cell. Optionally, Sall4 (Zhang et al., Nat Cell Biol.8(10):1114-23 (2006)) can be expressed in pace of any or all of Myc,Klf4, and Sox2.

Efficiency of induction to pluripotency as described herein can befurther improved by inclusion in non-pluripotent cells of, e.g., one ormore of, UTF1, SV40, TERT (either by introduction of an expressioncassette encoding these gene products, or by contacting the cells withthe proteins themselves) and/or by reducing expression of p53 (e.g., bysiRNA). See, e.g., Zhao, et al., Cell Stem Cell 3:475-479 (2008).

B. Transcription Factor Proteins

As detailed herein, a number of embodiments of the invention involvesintroduction of one or more polypeptides into cells, thereby inducingpluripotency in the cell. As discussed above, introduction of apolypeptide into a cell can comprise introduction of a polynucleotidecomprising one or more expression cassettes into a cell and inducingexpression, thereby introducing the polypeptides into the cell bytranscription and translation from the expression cassette.Alternatively, one can introduce an exogenous polypeptide (i.e., aprotein provided from outside the cell and/or that is not produced bythe cell) into the cell by a number of different methods that do notinvolve introduction of a polynucleotide encoding the polypeptide.

Accordingly, for any embodiment of the invention described hereinreferring either to introduction of a polypeptide into a cell, orintroduction of an expression cassette encoding a polypeptide into acell, it should be understood that the present invention also expresslyprovides for exogenous introduction of the polypeptide as a protein intothe cell. Therefore, in some embodiments, mammalian non-pluripotentcells are induced to pluripotency by a) exogenously introducing one ormore of a Klf polypeptide, an Oct polypeptide, a Myc polypeptide, and/ora Sox polypeptide into the non-pluripotent cells; and optionally b)contacting the cells with one or more of a MEK inhibitor, an agent thatinhibits H3K9 methylation, an L-type Ca channel agonist; an activator ofthe cAMP pathway; a DNA methyltransferase (DNMT) inhibitor; a nuclearreceptor ligand; a GSK3 inhibitor; a MEK inhibitor, a TGFβ receptor/ALK5inhibitor, a HDAC inhibitor; or an Erk inhibitor, thereby producinginduced pluripotent stem cells.

In some embodiments, in some embodiments of the invention,non-pluripotent cells are identified that endogenously express at leastone (and optionally, two or three of) proteins from the group consistingof a Oct polypeptide, a Klf polypeptide, a Myc polypeptide, and a Soxpolypeptide. The remaining (non-endogenously expressed) proteins fromthe group can then be exogenously introduced in the cells, and screenedfor re-programming and/or dedifferentiation into pluripotent cells,optionally in the presence of one or more of a MEK inhibitor, an agentthat inhibits H3K9 methylation, an L-type Ca channel agonist; anactivator of the cAMP pathway; a DNA methyltransferase (DNMT) inhibitor;a nuclear receptor ligand; a GSK3 inhibitor; a MEK inhibitor, a TGFβreceptor/ALK5 inhibitor, a HDAC inhibitor; or an Erk inhibitor.

In some embodiments, cells are identified that endogenously express aSox polypeptide and/or a Myc polypeptide, and as a second step an Octpolypeptide and a Klf polypeptide is exogenously introduced in the cell,thereby inducing conversion of the cell into a pluripotent cell. In someembodiments, cells are identified that endogenously express an Octpolypeptide and/or a Klf polypeptide and/or a Myc polypeptide, and a Soxpolypeptide is exogenously introduced in the cell, thereby inducingconversion of the cell into a pluripotent cell.

Exogenous introduction of a polypeptide into a cell can occur in anynumber of ways. One or more proteins can simply be cultured in thepresence of target cells under conditions to allow for introduction ofthe proteins into the cell. In some embodiments, the exogenous proteinscomprise the transcription factor polypeptide of interest linked (e.g.,linked as a fusion protein or otherwise covalently or non-covalentlylinked) to a polypeptide that enhances the ability of the transcriptionfactor to enter the cell (and optionally the cell nucleus).

Examples of polypeptide sequences that enhance transport acrossmembranes include, but are not limited to, the Drosophila homeoproteinantennapedia transcription protein (AntHD) (Joliot et al., New Biol. 3:1121-34, 1991; Joliot et al., Proc. Natl. Acad. Sci. USA, 88: 1864-8,1991; Le Roux et al., Proc. Natl. Acad. Sci. USA, 90: 9120-4, 1993), theherpes simplex virus structural protein VP22 (Elliott and O'Hare, Cell88: 223-33, 1997); the HIV-1 transcriptional activator TAT protein(Green and Loewenstein, Cell 55: 1179-1188, 1988; Frankel and Pabo, Cell55: 1 289-1193, 1988); delivery enhancing transporters such as describedin U.S. Pat. No. 6,730,293 (including but not limited to an peptidesequence comprising at least 7-25 contiguous arginines); andcommercially available Penetratin™ 1 peptide, and the Diatos PeptideVectors (“DPVs”) of the Vectocell® platform available from Daitos S. A.of Paris, France. See also, WO/2005/084158 and WO/2007/123667 andadditional transporters described therein. Not only can these proteinspass through the plasma membrane but the attachment of other proteins,such as the transcription factors described herein, is sufficient tostimulate the cellular uptake of these complexes.

In some embodiments, the transcription factor polypeptides describedherein are exogenously introduced as part of a liposome, or lipidcocktail such as commercially available Fugene6 and Lipofectamine). Inanother alternative, the transcription factor proteins can bemicroinjected or otherwise directly introduced into the target cell.

As discussed in the Examples, the inventors have found that incubationof cells with the transcription factor polypeptides of the invention forextended periods is toxic to the cells. Therefore, the present inventionprovides for intermittent incubation of non-pluripotent mammalian cellswith one or more of Klf polypeptide, an Oct polypeptide, a Mycpolypeptide, and/or a Sox polypeptide, with intervening periods ofincubation of the cells in the absence of the one or more polypeptides.In some embodiments, the cycle of incubation with and without thepolypeptides can be repeated for 2, 3, 4, 5, 6, or more times and isperformed for sufficient lengths of time (i.e., the incubations with andwithout proteins) to achieve the development of pluripotent cells.Various agents (e.g., MEK inhibitor and/or GSK inhibitor and/or TGFbetainhibitor) can be included to improve efficiency of the method.

C. Small Molecule Replacement of iPS Transcription Factors

In some embodiments of the invention, a cell expresses (endogenously orheterologously) at least one protein selected from an Oct polypeptide, aKlf polypeptide, a Myc polypeptide, and a Sox polypeptide (e.g., atleast 1, 2, or 3 of these) and is contacted with at least one agent,wherein the agent is sufficient to result in induction of the cell intoa pluripotent stem cell in the absence of expression of one or more ofthe remaining non-expressed proteins, i.e., any of the Oct polypeptide,the Klf polypeptide, the Myc polypeptide, or the Sox polypeptide notexpressed in the cell.

As shown in the Examples, contacting the cell with certain agents can“complement” or replace what is generally otherwise understood as anecessary expression of one of these proteins to result in pluripotentcells. By contacting a cell with an agent that functionally replacesexpression of one of the proteins listed above, it is possible togenerate pluripotent cells with expression of all of the above-listedproteins except the protein replaced or complemented by the agent. Theremaining proteins can be expressed endogenously, heterologously, or insome combination of the two (for example, a Sox and Myc polypeptidecould be expressed endogenously, a Klf polypeptide could be expressedheterologously and the Oct polypeptide could be “replaced” by insteadcontacting the cell with a complementary agent such as an agent thatinhibits methylation of H3K9 or promotes H3K9 demethylation).

Further, small molecules can improve the efficiency of a process forgenerating pluripotent cells (e.g., iPS cells). For example, improvedefficiency can be manifested by speeding the time to generate suchpluripotent cells (e.g., by shortening the time to development ofpluripotent cells by at least a day compared to a similar or sameprocess without the small molecule). Alternatively, or in combination, asmall molecule can increase the number of pluripotent cells generated bya particular process (e.g., increasing the number in a given time periodby at least 10%, 50%, 100%, 200%, 500%, etc. compared to a similar orsame process without the small molecule).

As described in the Examples, cells that heterologously express Klf4,Sox2, and Myc can be induced to pluripotency by further contacting thecells with an agent that inhibits H3K9 methylation without heterologousexpression of Oct4. Indeed, it has been found that pluripotency can beinduced by contacting non-pluripotent cells with an agent that inhibitsH3K9 methylation and introduction of either Oct 4 alone (e.g., withoutalso introducing a vector for expressing a Myc polypeptide, a Soxpolypeptide, or a KLf polypeptide) or Oct4 with Klf4. Cells that can beinduced to pluripotency include, but are not limited to, neuralprogenitor cells and fibroblasts. Agents that inhibit H3K9 methylationinclude agents that inhibit methylases (also known as methyltransferases) that target H3K9. For example, G9a histonemethyltransferase methylates H3K9 and inhibition of G9a histonemethyltransferase is known to reduce methylation of H3K9. See, e.g.,Kubicek, et al., Mol. Cell. 473-481 (2007). An example of a G9a histonemethyltransferase useful according to the methods of the invention isBIX01294 (see, e.g., Kubicek, et al., Mol. Cell 473-481 (2007)), orsalts, hydrates, isoforms, racemates, solvates and prodrug formsthereof. Bix01294 is displayed below:

The Bix01294 compounds of the present invention also include the salts,hydrates, solvates and prodrug forms. Bix01294 possesses asymmetriccarbon atoms (optical centers) or double bonds; the racemates,diastereomers, geometric isomers and individual isomers are all intendedto be encompassed within the scope of the present invention. Forexample, the compound of the present invention can be the R-isomer orthe S-isomer, or a mixture thereof. In addition, the compound of thepresent invention can be the E-isomer or the Z-isomer, or a combinationthereof.

In some embodiments, the agent that inhibits H3K9 methylation is asubstrate analog of a histone methyl transferase. The substrate of anumber of methyl transferases is S-adenosyl-methionine (SAM). Thus, insome embodiments, the agent that inhibits H3K9 methylation is a SAManalog. Exemplary SAM analogs include, but are not limited to,methylthio-adenosine (MTA), sinefungin, and S-adenosyl-homocysteine(SAH). In other embodiments, the agent that inhibits H3K9 methylationdoes not compete with SAM on a histone methyl transferase.

The resulting pluripotent cells (from heterologous expression and/orsmall molecule “replacement”) can develop into many or all of the threemajor tissue types: endoderm (e.g., interior gut lining), mesoderm(e.g., muscle, bone, blood), and ectoderm (e.g., epidermal tissues andnervous system), but, optionally, may show restrictions to theirdevelopmental potential (e.g., they may not form placental tissue, orother cell types of a defined lineage). The cells can be human ornon-human (e.g., primate, rat, mouse, rabbit, bovine, dog, cat, pig,etc.).

In other embodiments, BIX01294, or other agents that inhibit H3K9methylation or promote H3K9 demethylation can be used to inducepluripotency in cells that were previously not pluripotent. In someembodiments, an agent that inhibits H3K9 methylation is used to induceOct4 expression in cells, or at least alterations in Oct4 promoter DNAmethylation and/or histone methylation to allow for induction of cellsinto pluripotency. Thus, in some embodiments, cells that are notinitially pluripotent cells are contacted with an agent that inhibitsH3K9 methylation to induce the cells to become pluripotent. Indeed,without intending to limit the scope of the invention to a particularmode of action, the inventors believe that contacting non-pluripotentcells with an agent that inhibits H3K9 methylation or promotes H3K9demethylation will improve any method of inducing cells to pluripotency.For example, the agent that inhibits H3K9 methylation can be contactedto a non-pluripotent cell and induced to pluripotency in a methodcomprising contacting the non-pluripotent cells with an agent thatinhibits H3K9 methylation, optionally also contacting the cells with oneor more of an L-type Ca channel agonist; an activator of the cAMPpathway; a DNA methyltransferase (DNMT) inhibitor; a nuclear receptorligand; a GSK3 inhibitor; a MEK inhibitor, a TGFβ receptor/ALK5inhibitor, a HDAC inhibitor; or an Erk inhibitor, wherein each compoundis included in an amount sufficient in improve the efficiency ofinduction. In some embodiments as described in this paragraph, Oct4only, or Oct4/Klf4, or Sox2/Klf4 are further heterologously expressed inthe non-pluripotent cells resulting in induction of pluripotencyfollowing contacting with agents as described herein.

The inventors have also found that the combination of a GSK inhibitorand an HDAC inhibitor, or a GSK inhibitor and a cAMP pathway activator,or a GSK inhibitor and an ALK5 inhibitor can induce to pluripotency anyof mouse or human fibroblasts or keratinocytes that express any of: Oct4alone, Oct4/Klf4 or Sox2/Klf4 (data not shown). In other embodiments, anon-pluripotent cell is induced to pluripotency in a method comprisingcontacting the non-pluripotent cells with a GSK3 inhibitor, optionallyalso contacting the cells with one or more of an L-type Ca channelagonist; an activator of the cAMP pathway; a DNA methyltransferase(DNMT) inhibitor; a nuclear receptor ligand; a GSK3 inhibitor; a MEKinhibitor, a TGFβ receptor/ALK5 inhibitor, a HDAC inhibitor; or an Erkinhibitor, wherein each compound is included in an amount sufficient inimprove the efficiency of induction. In other embodiments, anon-pluripotent cell is induced to pluripotency in a method comprisingcontacting the non-pluripotent cells with a TGFβ receptor/ALK5inhibitor, optionally also contacting the cells with one or more of anL-type Ca channel agonist; an activator of the cAMP pathway; a DNAmethyltransferase (DNMT) inhibitor; a nuclear receptor ligand; a GSK3inhibitor; a MEK inhibitor, a HDAC inhibitor; or an Erk inhibitor,wherein each compound is included in an amount sufficient in improve theefficiency of induction. In other embodiments, a non-pluripotent cell isinduced to pluripotency in a method comprising contacting thenon-pluripotent cells with a HDAC inhibitor, optionally also contactingthe cells with one or more of an L-type Ca channel agonist; an activatorof the cAMP pathway; a DNA methyltransferase (DNMT) inhibitor; a nuclearreceptor ligand; a GSK3 inhibitor; a MEK inhibitor, a TGFβ receptor/ALK5inhibitor, or an Erk inhibitor, wherein each compound is included in anamount sufficient in improve the efficiency of induction. In otherembodiments, a non-pluripotent cell is induced to pluripotency in amethod comprising contacting the non-pluripotent cells with a MEKinhibitor, optionally also contacting the cells with one or more of anL-type Ca channel agonist; an activator of the cAMP pathway; a DNAmethyltransferase (DNMT) inhibitor; a nuclear receptor ligand; a GSK3inhibitor; a TGFβ receptor/ALK5 inhibitor, a HDAC inhibitor; or an Erkinhibitor, wherein each compound is included in an amount sufficient inimprove the efficiency of induction. In other embodiments, anon-pluripotent cell is induced to pluripotency in a method comprisingcontacting the non-pluripotent cells with two, three, four, five, six,seven, eight, nine, or each of a MEK inhibitor, an L-type Ca channelagonist; an agent that inhibits H3K9 methylation, an activator of thecAMP pathway; a DNA methyltransferase (DNMT) inhibitor; a nuclearreceptor ligand; a GSK3 inhibitor; a TGFβ receptor/ALK5 inhibitor, aHDAC inhibitor; or an Erk inhibitor, wherein each compound is includedin an amount sufficient in improve the efficiency of induction. In someembodiments as described in this paragraph, Oct4 only, or Oct4/Klf4, orSox2/Klf4 are further heterologously expressed in the non-pluripotentcells resulting in induction of pluripotency following contacting withagents as described herein.

Exemplary L-type calcium channel agonists include, but are not limitedto, BayK8644 (see, e.g., Schramm, et al., Nature 303:535-537 (1983)),Dehydrodidemnin B (see, e.g., U.S. Pat. No. 6,030,943), FPL 64176 (FPL)(see, e.g., Liwang, et al., Neuropharmacology 45:281-292 (2003)),S(+)-PN 202-791 (see, e.g., Kennedy, et al., Neuroscience 49:937-44(1992)) and CGP 48506 (see, e.g., Chahine, et al., Canadian Journal ofPhysiology and Pharmacology 81:135-141 (2003)).

Exemplary activators of the cAMP pathway include, but are not limitedto, forskolin (see, e.g., Liang, et al., Endocrinology 146: 4437-4444(2005)), FSH (see, Liang, supra), milrinone (see, Liang, supra),cilostamide (see, Liang, supra), rolipram (see, Liang, supra), dbcAMP(see, Liang, supra) and 8-Br-cAMP (see, Liang, supra).

Exemplary DNA methyltransferase (DNMT) inhibitors can include antibodiesthat bind, dominant negative variants of, and siRNA and antisensenucleic acids that suppress expression of DNMT. DNMT inhibitors include,but are not limited to, RG108 (available, e.g., from Sigma-Aldrich),5-aza-C (5-azacitidine or azacitidine) (see, e.g., Schermelleh, et al.,Nature Methods 2:751-6 (2005)), 5-aza-2′-deoxycytidine (5-aza-CdR) (see,e.g., Zhu, Clinical Medicinal Chemistry 3(3):187-199 (2003)), decitabine(see, e.g., Gore, Nature Clinical Practice Oncology 2:S30-S35 (2005)),doxorubicin (see, e.g., Levenson, Molecular Pharmacology 71:635-637(2007)), EGCG ((−)-epigallocatechin-3-gallate) (see, e.g., Fang, et al.,Cancer Research 63:7563-7570 (2003)), RG108 (see, e.g., Carninci, etal., WO2008/126932, incorporated herein by reference) and zebularine(see, Carninci, supra).

Exemplary nuclear receptor ligands, i.e., agonists, antagonists,activators and/or repressors of nuclear receptors, can modulate localgene expression or transcription at the site of delivery. Nuclearreceptor agonist (and also nuclear receptor antagonists) can be used. Insome embodiments, nuclear receptors are co-regulators of transcription.Activation or inhibition of certain nuclear receptors regulateepigenetic states of specific gene loci where they bind. The inventorshave found that dexamethasone (e.g., at 1 μM, a glucocorticoid receptoragonist), ciglitazone and Fmoc-Leu (both used at 5 μM) (a PPAR agonist),and Bexarotene (e.g., at (3 μM) (a RXR antagonist) can enhance cellularreprogramming. Representative nuclear receptor ligands include, but arenot limited to, estradiol (e.g., 17-beta estradiol), all-trans retinoicacid, 13-cis retinoic acid, dexamethasone, clobetasol, androgens,thyroxine, vitamin D3 glitazones, troglitazone, pioglitazone,rosiglitazone, prostaglandins, and fibrates (e.g., bezafibrate,ciprofibrate, gemfibrozil, fenofibrate and clofibrate). Furthermore, theactivity of endogenous ligands (such as the hormones estradiol andtestosterone) when bound to their cognate nuclear receptors is normallyto upregulate gene expression. This upregulation or stimulation of geneexpression by the ligand can be referred to as an agonist response. Theagonistic effects of endogenous hormones can also be mimicked by certainsynthetic ligands, for example, the glucocortocoid receptoranti-inflammatory drug dexamethasone. Agonist ligands function byinducing a conformation of the receptor which favors coactivatorbinding. (See, e.g., WO08011093A incorporate herein by reference.)

Inhibitors of GSK3 can include antibodies that bind, dominant negativevariants of, and siRNA and antisense nucleic acids that target GSK3.Specific examples of GSK3inhibitors include, but are not limited to,Kenpaullone, 1-Azakenpaullone, CHIR99021, CHIR98014, AR-A014418 (see,e.g., Gould, et al., The International Journal ofNeuropsychopharmacology 7:387-390 (2004)), CT 99021 (see, e.g., Wagman,Current Pharmaceutical Design 10:1105-1137 (2004)), CT 20026 (see,Wagman, supra), SB216763 (see, e.g., Martin, et al., Nature Immunology6:777-784 (2005)), AR-A014418 (see, e.g., Noble, et al., PNAS102:6990-6995 (2005)), lithium (see, e.g., Gould, et al.,Pharmacological Research 48: 49-53 (2003)), SB 415286 (see, e.g., Frame,et al., Biochemical Journal 359:1-16 (2001)) and TDZD-8 (see, e.g.,Chin, et al., Molecular Brain Research, 137(1-2):193-201 (2005)).Further exemplary GSK3 inhibitors available from Calbiochem (see, e.g.,Dalton, et al., WO2008/094597, herein incorporated by reference),include but are not limited to BIO (2′Z,3′£)-6-Bromoindirubin-3′-oxime(GSK3 Inhibitor IX); BIO-Acetoxime(2′Z,3′E)-6-Bromoindirubin-3′-acetoxime (GSK3 Inhibitor X);(5-Methyl-1H-pyrazol-3-yl)-(2-phenylquinazolin-4-yl)amine(GSK3-Inhibitor XIII); Pyridocarbazole-cyclopenadienylruthenium complex(GSK3 Inhibitor XV); TDZD-84-Benzyl-2-methyl-1,2,4-thiadiazolidine-3,5-dione (GSK3beta InhibitorI); 2-Thio(3-iodobenzyl)-5-(1-pyridyl)-[1,3,4]-oxadiazole (GSK3betaInhibitor II); OTDZT 2,4-Dibenzyl-5-oxothiadiazolidine-3-thione(GSK3beta Inhibitor III); alpha-4-Dibromoacetophenone (GSK3betaInhibitor VII); AR-AO 14418N-(4-Methoxybenzyl)-N′-(5-nitro-1,3-thiazol-2-yl)urea (GSK-3betaInhibitor VIII);3-(1-(3-Hydroxypropyl)-1H-pyrrolo[2,3-b]pyridin-3-yl]-4-pyrazin-2-yl-pyrrole-2,5-dione(GSK-3beta Inhibitor XI); TWSI 19 pyrrolopyrimidine compound (GSK3betaInhibitor XII); L803 H-KEAPPAPPQSpP-NH2 (SEQ ID NO:1) or itsMyristoylated form (GSK3beta Inhibitor XIII);2-Chloro-1-(4,5-dibromo-thiophen-2-yl)-ethanone (GSK3beta Inhibitor VI);AR-AO144-18; SB216763; and SB415286. Residues of GSK3b that interactwith inhibitors have been identified. See, e.g., Bertrand et al., J. MolBiol. 333(2): 393-407 (2003). GSK3 inhibitors can activate, for example,the Wnt/β-catenin pathway. Many of β-catenin downstream genesco-regulate pluripotency gene networks. For example, a GSK inhibitoractivates cMyc expression as well as enhances its protein stability andtranscriptional activity. Thus, in some embodiments, GSK3 inhibitors canbe used to stimulate endogenous Myc polypeptide expression in a cell,thereby eliminating the need for Myc expression to induce pluripotency.

Inhibitors of MEK can include antibodies to, dominant negative variantsof, and siRNA and antisense nucleic acids that suppress expression of,MEK. Specific examples of MEK inhibitors include, but are not limitedto, PD0325901, (see, e.g., Rinehart, et al., Journal of ClinicalOncology 22: 4456-4462 (2004)), PD98059 (available, e.g., from CellSignaling Technology), U0126 (available, for example, from CellSignaling Technology), SL 327 (available, e.g., from Sigma-Aldrich),ARRY-162 (available, e.g., from Array Biopharma), PD184161 (see, e.g.,Klein, et al., Neoplasia 8:1-8 (2006)), PD184352 (CI-1040) (see, e.g.,Mattingly, et al., The Journal of Pharmacology and ExperimentalTherapeutics 316:456-465 (2006)), sunitinib (see, e.g., Voss, et al.,US2008004287 incorporated herein by reference), sorafenib (see, Vosssupra), Vandetanib (see, Voss supra), pazopanib (see, e.g., Voss supra),Axitinib (see, Voss supra) and PTK787 (see, Voss supra).

Currently, several MEK inhibitors are undergoing clinical trialevaluations. CI-1040 has been evaluate in Phase I and II clinical trialsfor cancer (see, e.g., Rinehart, et al., Journal of Clinical Oncology22(22):4456-4462 (2004)). Other MEK inhibitors being evaluated inclinical trials include PD184352 (see, e.g., English, et al., Trends inPharmaceutical Sciences 23(1):40-45 (2002)), BAY 43-9006 (see, e.g.,Chow, et al., Cytometry (Communications in Clinical Cytometry) 46:72-78(2001)), PD-325901 (also PD0325901), GSK1120212, ARRY-438162, RDEA119,AZD6244 (also ARRY-142886 or ARRY-886), RO5126766, XL518 and AZD8330(also ARRY-704). (See, e.g., information from the National Institutes ofHealth located on the World Wide Web at clinicaltrials.gov as well asinformation from the Nation Cancer Institute located on the World WideWeb at cancer.gov/clinicaltrials.

TGF beta receptor (e.g., ALK5) inhibitors can include antibodies to,dominant negative variants of, and antisense nucleic acids that suppressexpression of, TGF beta receptors (e.g., ALK5). Exemplary TGFβreceptor/ALK5 inhibitors include, but are not limited to, SB431542 (see,e.g., Inman, et al., Molecular Pharmacology 62(1):65-74 (2002)),A-83-01, also known as3-(6-Methyl-2-pyridinyl)-N-phenyl-4-(4-quinolinyl)-1H-pyrazole-1-carbothioamide(see, e.g., Tojo, et al., Cancer Science 96(11):791-800 (2005), andcommercially available from, e.g., Toicris Bioscience);2-(3-(6-Methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine,Wnt3a/BIO (see, e.g., Dalton, et al., WO2008/094597, herein incorporatedby reference), BMP4 (see, Dalton, supra), GW788388(-{4-[3-(pyridin-2-yl)-1H-pyrazol-4-yl]pyridin-2-yl}-N-(tetrahydro-2H-pyran-4-yl)benzamide)(see, e.g., Gellibert, et al., Journal of Medicinal Chemistry49(7):2210-2221 (2006)), SM16 (see, e.g., Suzuki, et al., CancerResearch 67(5):2351-2359 (2007)), IN-1130(3-((5-(6-methylpyridin-2-yl)-4-(quinoxalin-6-yl)-1H-imidazol-2-yl)methyl)benzamide)(see, e.g., Kim, et al., Xenobiotica 38(3):325-339 (2008)), GW6604(2-phenyl-4-(3-pyridin-2-yl-1H-pyrazol-4-yl)pyridine) (see, e.g., deGouville, et al., Drug News Perspective 19(2):85-90 (2006)), SB-505124(2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridinehydrochloride) (see, e.g., DaCosta, et al., Molecular Pharmacology65(3):744-752 (2004)) and pyrimidine derivatives (see, e.g., thoselisted in Stiefl, et al., WO2008/006583, herein incorporated byreference). Further, while “an ALK5 inhibitor” is not intended toencompass non-specific kinase inhibitors, an “ALK5 inhibitor” should beunderstood to encompass inhibitors that inhibit ALK4 and/or ALK7 inaddition to ALK5, such as, for example, SB-431542 (see, e.g., Inman, etal., J, Mol. Pharmacol. 62(1): 65-74 (2002). Without intending to limitthe scope of the invention, it is believed that ALK5 inhibitors affectthe mesenchymal to epithelial conversion/transition (MET) process.TGFβ/activin pathway is a driver for epithelial to mesenchymaltransition (EMT). Therefore, inhibiting the TGFβ/activin pathway canfacilitate MET (i.e. reprogramming) process.

In view of the data herein showing the effect of inhibiting ALK5, it isbelieved that inhibition of the TGFβ/activin pathway will have similareffects. Thus, any inhibitor (e.g., upstream or downstream) of theTGFβ/activin pathway can be used in combination with, or instead of,ALK5 inhibitors as described in each paragraph herein. ExemplaryTGFβ/activin pathway inhibitors include but are not limited to: TGF betareceptor inhibitors, inhibitors of SMAD 2/3 phosphorylation, inhibitorsof the interaction of SMAD 2/3 and SMAD 4, and activators/agonists ofSMAD 6 and SMAD 7. Furthermore, the categorizations described below aremerely for organizational purposes and one of skill in the art wouldknow that compounds can affect one or more points within a pathway, andthus compounds may function in more than one of the defined categories.

TGF beta receptor inhibitors can include antibodies to, dominantnegative variants of and siRNA or antisense nucleic acids that targetTGF beta receptors. Specific examples of inhibitors include but are notlimited to SU5416;2-(5-benzo[1,3]dioxol-5-yl-2-tert-butyl-3H-imidazol-4-yl)-6-methylpyridinehydrochloride (SB-505124); lerdelimumb (CAT-152); metelimumab (CAT-192);GC-1008; ID11; AP-12009; AP-11014; LY550410; LY580276; LY364947;LY2109761; SB-505124; SB-431542; SD-208; SM16; NPC-30345; Ki26894;SB-203580; SD-093; Gleevec; 3,5,7,2′,4′-pentahydroxyflavone (Morin);activin-M108A; P144; soluble TBR2-Fc; and antisense transfected tumorcells that target TGF beta receptors. (See, e.g., Wrzesinski, et al.,Clinical Cancer Research 13(18):5262-5270 (2007); Kaminska, et al., ActaBiochimica Polonica 52(2):329-337 (2005); and Chang, et al., Frontiersin Bioscience 12:4393-4401 (2007).)

Inhibitors of SMAD 2/3 phosphorylation can include antibodies to,dominant negative variants of and antisense nucleic acids that targetSMAD2 or SMAD3. Specific examples of inhibitors include PD169316;SB203580; SB-431542; LY364947; A77-01; and3,5,7,2′,4′-pentahydroxyflavone (Morin). (See, e.g., Wrzesinski, supra;Kaminska, supra; Shimanuki, et al., Oncogene 26:3311-3320 (2007); andKataoka, et al., EP1992360, incorporated herein by reference.)

Inhibitors of the interaction of SMAD 2/3 and smad4 can includeantibodies to, dominant negative variants of and antisense nucleic acidsthat target SMAD2, SMAD3 and/or smad4. Specific examples of inhibitorsof the interaction of SMAD 2/3 and SMAD4 include but are not limited toTrx-SARA, Trx-xFoxHlb and Trx-Lef1. (See, e.g., Cui, et al., Oncogene24:3864-3874 (2005) and Zhao, et al., Molecular Biology of the Cell,17:3819-3831 (2006).)

Activators/agonists of SMAD 6 and SMAD 7 include but are not limited toantibodies to, dominant negative variants of and antisense nucleic acidsthat target SMAD 6 or SMAD 7. Specific examples of inhibitors includebut are not limited to smad7-as PTO-oligonucleotides. (See, e.g.,Miyazono, et al., U.S. Pat. No. 6,534,476, and Steinbrecher, et al.,US2005119203, both incorporated herein by reference.)

Exemplary HDAC inhibitors can include antibodies that bind, dominantnegative variants of, and siRNA and antisense nucleic acids that targetHDAC. HDAC inhibitors include, but are not limited to, TSA (trichostatinA) (see, e.g., Adcock, British Journal of Pharmacology 150:829-831(2007)), VPA (valproic acid) (see, e.g., Munster, et al., Journal ofClinical Oncology 25:18 S (2007): 1065), sodium butyrate (NaBu) (see,e.g., Han, et al., Immunology Letters 108:143-150 (2007)), SAHA(suberoylanilide hydroxamic acid or vorinostat) (see, e.g., Kelly, etal., Nature Clinical Practice Oncology 2:150-157 (2005)), sodiumphenylbutyrate (see, e.g., Gore, et al., Cancer Research 66:6361-6369(2006)), depsipeptide (FR901228, FK228) (see, e.g., Zhu, et al., CurrentMedicinal Chemistry 3(3):187-199 (2003)), trapoxin (TPX) (see, e.g.,Furumai, et al., PNAS 98(1):87-92 (2001)), cyclic hydroxamicacid-containing peptide 1 (CHAP1) (see, Furumai supra), MS-275 (see,e.g., Carninci, et al., WO2008/126932, incorporated herein byreference)), LBH589 (see, e.g., Goh, et al., WO2008/108741 incorporatedherein by reference) and PXD101 (see, Goh, supra). In general at theglobal level, pluripotent cells have more histone acetylation, anddifferentiated cells have less histone acetylation. Histone acetylationis also involved in histone and DNA methylation regulation. In someembodiments, HDAC inhibitors facilitate activation of silencedpluripotency genes.

Exemplary ERK inhibitors include PD98059 (see, e.g., Zhu, et al.,Oncogene 23:4984-4992 (2004)), U0126 (see, Zhu, supra), FR180204 (see,e.g., Ohori, Drug News Perspective 21(5):245-250 (2008)), sunitinib(see, e.g., Ma, et al., US2008004287 incorporated herein by reference),sorafenib (see, Ma, supra), Vandetanib (see, Ma, supra), pazopanib (see,Ma, supra), Axitinib (see, Ma, supra) and PTK787 (see, Ma, supra).

Once expression cassettes have been introduced into the cells and/or thecells have been contacted with the one or more agents, the cells can beoptionally screen for characteristics of pluripotent stem cells, therebyidentifying those cells in a mixture that are pluripotent. Such cellscan be, for example, isolated from the other cells and used further asappropriate.

III. Non-Pluripotent Cells

As used herein, “non-pluripotent cells” refer to mammalian cells thatare not pluripotent cells. Examples of such cells include differentiatedcells as well as progenitor cells. Examples of differentiated cellsinclude, but are not limited to, cells from a tissue selected from bonemarrow, skin, skeletal muscle, fat tissue and peripheral blood.Exemplary cell types include, but are not limited to, fibroblasts,hepatocytes, myoblasts, neurons, osteoblasts, osteoclasts, and T-cells.

In some embodiments where an individual is to be treated with theresulting pluripotent cells, the individual's own non-pluripotent cellsare used to generate pluripotent cells according to the methods of theinvention.

Cells can be from, e.g., humans or non-human mammals. Exemplarynon-human mammals include, but are not limited to, mice, rats, cats,dogs, rabbits, guinea pigs, hamsters, sheep, pigs, horses, and bovines.

IV. Transformation

This invention relies on routine techniques in the field of recombinantgenetics. Basic texts disclosing the general methods of use in thisinvention include Sambrook et al., Molecular Cloning, A LaboratoryManual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: ALaboratory Manual (1990); and Current Protocols in Molecular Biology(Ausubel et al., eds., 1994)).

In some embodiments, the species of cell and protein to be expressed isthe same. For example, if a mouse cell is used, a mouse ortholog isintroduced into the cell. If a human cell is used, a human ortholog isintroduced into the cell.

It will be appreciated that where two or more proteins are to beexpressed in a cell, one or multiple expression cassettes can be used.For example, where one expression cassette is to express multiplepolypeptides, a polycistronic expression cassette can be used.

A. Plasmid Vectors

In certain embodiments, a plasmid vector is contemplated for use totransform a host cell. In general, plasmid vectors containing repliconand control sequences which are derived from species compatible with thehost cell are used in connection with these hosts. The vector can carrya replication site, as well as marking sequences which are capable ofproviding phenotypic selection in transformed cells.

B. Viral Vectors

The ability of certain viruses to infect cells or enter cells viareceptor-mediated endocytosis, and to integrate into host cell genomeand express viral genes stably and efficiently have made them attractivecandidates for the transfer of foreign nucleic acids into cells (e.g.,mammalian cells). Non-limiting examples of virus vectors that may beused to deliver a nucleic acid of the present invention are describedbelow.

i. Adenoviral Vectors

A particular method for delivery of the nucleic acid involves the use ofan adenovirus expression vector. Although adenovirus vectors are knownto have a low capacity for integration into genomic DNA, this feature iscounterbalanced by the high efficiency of gene transfer afforded bythese vectors. “Adenovirus expression vector” is meant to include thoseconstructs containing adenovirus sequences sufficient to (a) supportpackaging of the construct and (b) to ultimately express a tissue orcell-specific construct that has been cloned therein. Knowledge of thegenetic organization or adenovirus, a ˜36 kb, linear, double-strandedDNA virus, allows substitution of large pieces of adenoviral DNA withforeign sequences up to 7 kb (Grunhaus et al., Seminar in Virology,200(2):535-546, 1992)).

ii. AAV Vectors

The nucleic acid may be introduced into the cell using adenovirusassisted transfection. Increased transfection efficiencies have beenreported in cell systems using adenovirus coupled systems (Kelleher andVos, Biotechniques, 17(6):1110-7, 1994; Cotten et al., Proc Natl AcadSci USA, 89(13):6094-6098, 1992; Curiel, Nat Immun, 13(2-3):141-64,1994.). Adeno-associated virus (AAV) is an attractive vector system asit has a high frequency of integration and it can infect non-dividingcells, thus making it useful for delivery of genes into mammalian cells,for example, in tissue culture (Muzyczka, Curr Top Microbiol Immunol,158:97-129, 1992) or in vivo. Details concerning the generation and useof rAAV vectors are described in U.S. Pat. Nos. 5,139,941 and 4,797,368,each incorporated herein by reference.

iii. Retroviral Vectors

Retroviruses have promise as gene delivery vectors due to their abilityto integrate their genes into the host genome, transferring a largeamount of foreign genetic material, infecting a broad spectrum ofspecies and cell types and of being packaged in special cell-lines(Miller et al., Am. J. Clin. Oncol., 15(3):216-221, 1992).

In order to construct a retroviral vector, a nucleic acid (e.g., oneencoding gene of interest) is inserted into the viral genome in theplace of certain viral sequences to produce a virus that isreplication-defective. To produce virions, a packaging cell linecontaining the gag, pol, and env genes but without the LTR and packagingcomponents is constructed (Mann et al., Cell, 33:153-159, 1983). When arecombinant plasmid containing a cDNA, together with the retroviral LTRand packaging sequences is introduced into a special cell line (e.g., bycalcium phosphate precipitation for example), the packaging sequenceallows the RNA transcript of the recombinant plasmid to be packaged intoviral particles, which are then secreted into the culture media (Nicolasand Rubinstein, In: Vectors: A survey of molecular cloning vectors andtheir uses, Rodriguez and Denhardt, eds., Stoneham: Butterworth, pp.494-513, 1988; Temin, In: Gene Transfer, Kucherlapati (ed.), New York:Plenum Press, pp. 149-188, 1986; Mann et al., Cell, 33:153-159, 1983).The media containing the recombinant retroviruses is then collected,optionally concentrated, and used for gene transfer. Retroviral vectorsare able to infect a broad variety of cell types. However, integrationand stable expression typically involves the division of host cells(Paskind et al., Virology, 67:242-248, 1975).

Lentiviruses are complex retroviruses, which, in addition to the commonretroviral genes gag, pol, and env, contain other genes with regulatoryor structural function. Lentiviral vectors are well known in the art(see, for example, Naldini et al., Science, 272(5259):263-267, 1996;Zufferey et al., Nat Biotechnol, 15(9):871-875, 1997; Blomer et al., J.Virol., 71(9):6641-6649, 1997; U.S. Pat. Nos. 6,013,516 and 5,994,136).Some examples of lentivirus include the Human Immunodeficiency Viruses:HIV-1, HIV-2 and the Simian Immunodeficiency Virus: SIV. Lentiviralvectors have been generated by multiply attenuating the HIV virulencegenes, for example, the genes env, vif, vpr, vpu and nef are deletedmaking the vector biologically safe.

Recombinant lentiviral vectors are capable of infecting non-dividingcells and can be used for both in vivo and ex vivo gene transfer andexpression of nucleic acid sequences. For example, recombinantlentivirus capable of infecting a non-dividing cell wherein a suitablehost cell is transfected with two or more vectors carrying the packagingfunctions, namely gag, pol and env, as well as rev and tat is describedin U.S. Pat. No. 5,994,136, incorporated herein by reference. One maytarget the recombinant virus by linkage of the envelope protein with anantibody or a particular ligand for targeting to a receptor of aparticular cell-type. By inserting a sequence (including a regulatoryregion) of interest into the viral vector, along with another gene whichencodes the ligand for a receptor on a specific target cell, forexample, the vector is now target-specific.

iv. Delivery Using Modified Viruses

A nucleic acid to be delivered may be housed within an infective virusthat has been engineered to express a specific binding ligand. The virusparticle will thus bind specifically to the cognate receptors of thetarget cell and deliver the contents to the cell. A novel approachdesigned to allow specific targeting of retrovirus vectors was developedbased on the chemical modification of a retrovirus by the chemicaladdition of lactose residues to the viral envelope. This modificationcan permit the specific infection of hepatocytes via sialoglycoproteinreceptors.

Another approach to targeting of recombinant retroviruses was designedin which biotinylated antibodies against a retroviral envelope proteinand against a specific cell receptor were used. The antibodies werecoupled via the biotin components by using streptavidin (Roux et al.,Proc. Nat'l Acad. Sci. USA, 86:9079-9083, 1989). Using antibodiesagainst major histocompatibility complex class I and class II antigens,they demonstrated the infection of a variety of human cells that borethose surface antigens with an ecotropic virus in vitro (Roux et al.,1989).

C. Vector Delivery and Cell Transformation

Suitable methods for nucleic acid delivery for transformation of a cell,a tissue or an organism for use with the current invention are believedto include virtually any method by which a nucleic acid (e.g., DNA) canbe introduced into a cell, a tissue or an organism, as described hereinor as would be known to one of ordinary skill in the art. Such methodsinclude, but are not limited to, direct delivery of DNA such as by exvivo transfection (Wilson et al., Science, 244:1344-1346, 1989, Nabeland Baltimore, Nature 326:711-713, 1987), optionally with Fugene6(Roche) or Lipofecyamine (Invitrogen), by injection (U.S. Pat. Nos.5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932,5,656,610, 5,589,466 and 5,580,859, each incorporated herein byreference), including microinjection (Harland and Weintraub, J. CellBiol., 101:1094-1099, 1985; U.S. Pat. No. 5,789,215, incorporated hereinby reference); by electroporation (U.S. Pat. No. 5,384,253, incorporatedherein by reference; Tur-Kaspa et al., Mol. Cell. Biol., 6:716-718,1986; Potter et al., Proc. Nat'l Acad. Sci. USA, 81:7161-7165, 1984); bycalcium phosphate precipitation (Graham and Van Der Eb, Virology,52:456-467, 1973; Chen and Okayama, Mol. Cell. Biol., 7(8):2745-2752,1987; Rippe et al., Mol. Cell. Biol., 10:689-695, 1990); by usingDEAE-dextran followed by polyethylene glycol (Gopal, Mol. Cell. Biol.,5:1188-1190, 1985); by direct sonic loading (Fechheimer et al., Proc.Nat'l Acad. Sci. USA, 84:8463-8467, 1987); by liposome mediatedtransfection (Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190,1982; Fraley et al., Proc. Nat'l Acad. Sci. USA, 76:3348-3352, 1979;Nicolau et al., Methods Enzymol., 149:157-176, 1987; Wong et al., Gene,10:87-94, 1980; Kaneda et al., Science, 243:375-378, 1989; Kato et al.,J Biol. Chem., 266:3361-3364, 1991) and receptor-mediated transfection(Wu and Wu, Biochemistry, 27:887-892, 1988; Wu and Wu, J. Biol. Chem.,262:4429-4432, 1987); each incorporated herein by reference); and anycombination of such methods.

V. Culturing of Cells

Cells to be induced to pluripotency can be cultured according to anymethod known in the art. General guidelines can be found in, e.g.,Maherali, et al., Cell Stem Cell 3:595-605 (2008).

In some embodiments, the cells are cultured in contact with feedercells. Exemplary feeder cells include, but are not limited to fibroblastcells, e.g., mouse embryonic fibroblast (MEF) cells. Methods ofculturing cells on feeder cells is known in the art.

In some embodiments, the cells are cultured in the absence of feedercells. Cells, for example, can be attached directly to a solid culturesurface (e.g., a culture plate), e.g., via a molecular tether. Theinventors have found that culturing cells induced to pluripotency have amuch greater efficiency of induction to pluripotency (i.e., a greaterportion of cells achieve pluripotency) when the cells are attacheddirectly to the solid culturing surface compared the efficiency ofotherwise identically-treated cells that are cultured on feeder cells.Exemplary molecular tethers include, but are not limited to, matrigel,an extracellular matrix (ECM), ECM analogs, laminin, fibronectin, orcollagen. Those of skill in the art however will recognize that this isa non-limiting list and that other molecules can be used to attach cellsto a solid surface. Methods for initial attachment of the tethers to thesolid surface are known in the art.

As used in this “culturing” section, “cells to be induced topluripotency” are induced by any method in the art, including, but notlimited to the methods described in this application.

VI. Uses for Pluripotent Cells

The present invention allows for the further study and development ofstem cell technologies, including but not limited to, prophylactic ortherapeutic uses. For example, in some embodiments, cells of theinvention (either pluripotent cells or cells induced to differentiatealong a desired cell fate) are introduced into individuals in needthereof, including but not limited to, individuals in need ofregeneration of an organ, tissue, or cell type. In some embodiments, thecells are originally obtained in a biopsy from an individual; inducedinto pluripotency as described herein, optionally induced todifferentiate (for examples into a particular desired progenitor cell)and then transplanted back into the individual. In some embodiments, thecells are genetically modified prior to their introduction into theindividual.

In some embodiments, the pluripotent cells generated according to themethods of the invention are subsequently induced to form, for example,hematopoietic (stem/progenitor) cells, neural (stem/progenitor) cells(and optionally, more differentiated cells, such as subtype specificneurons, oligodendrocytes, etc), pancreatic cells (e.g., endocrineprogenitor cell or pancreatic hormone-expressing cells), hepatocytes,cardiovascular (stem/progenitor) cells (e.g., cardiomyocytes,endothelial cells, smooth muscle cells), retinal cells, etc.

A variety of methods are known for inducing differentiation ofpluripotent stem cells into desired cell types. A non-limiting list ofrecent patent publications describing methods for inducingdifferentiation of stem cells into various cell fates follows: U.S.Patent Publication No. 2007/0281355; 2007/0269412; 2007/0264709;2007/0259423; 2007/0254359; 2007/0196919; 2007/0172946; 2007/0141703;2007/0134215.

A variety of diseases may be ameliorated by introduction, and optionallytargeting, of pluripotent cells of the invention to a particular injuredtissue. Examples of disease resulting from tissue injury include, butare not limited to, neurodegeneration disease, cerebral infarction,obstructive vascular disease, myocardial infarction, cardiac failure,chronic obstructive lung disease, pulmonary emphysema, bronchitis,interstitial pulmonary disease, asthma, hepatitis B (liver damage),hepatitis C (liver damage), alcoholic hepatitis (liver damage), hepaticcirrhosis (liver damage), hepatic insufficiency (liver damage),pancreatitis, diabetes mellitus, Crohn disease, inflammatory colitis,IgA glomerulonephritis, glomerulonephritis, renal insufficiency,decubitus, burn, sutural wound, laceration, incised wound, bite wound,dermatitis, cicatricial keloid, keloid, diabetic ulcer, arterial ulcerand venous ulcer.

The polypeptides described herein (e.g., one or more of a Klfpolypeptide, an Oct polypeptide, a Myc polypeptide, and a Soxpolypeptide) are themselves useful therapeutic agents alone, or incombination as described herein. For example, the polypeptides, orcombinations thereof, are useful for reducing tissue damage and thus canbe administered to treat, ameliorate, or prevent tissue damage. In someembodiments, a compound of the invention is administered to anindividual having, or at risk of having tissue damage to an internalorgan. Internal organs include, but are not limited to, brain, pancreas,liver, intestine, lung, kidney, or heart, wounding, e.g., by burn orcut. For example, in some embodiments, the compounds of the inventionare effective in reducing infarction size in reperfusion followingischemia. Thus, a protein of the invention can be administered toindividuals at risk of having, having, or who have had, a stroke.Similarly, a protein of the invention can be administered to individualsat risk of having, having, or who have had, a heart attack or cardiacdamage.

The agents described herein (e.g., an agent that inhibits H3K9methylation; an L-type Ca channel agonist; an activator of the cAMPpathway; a DNA methyltransferase (DNMT) inhibitor; a nuclear receptorligand; a GSK3 inhibitor; a MEK inhibitor, a TGFβ receptor/ALK5inhibitor, a HDAC inhibitor; or an Erk inhibitor.) are also usefultherapeutic agents alone, or in combination with each other as describedherein. For example, the agents, or combinations thereof, are useful forreducing tissue damage and thus can be administered to treat,ameliorate, or prevent tissue damage. In some embodiments, an agent ofthe invention is administered to an individual having, or at risk ofhaving tissue damage to an internal organ. Internal organs include, butare not limited to, brain, pancreas, liver, intestine, lung, kidney, orheart, wounding, e.g., by burn or cut. For example, in some embodiments,the agents of the invention are effective in reducing infarction size inreperfusion following ischemia. Thus, an agent of the invention can beadministered to individuals at risk of having, having, or who have had,a stroke. Similarly, an agent of the invention can be administered toindividuals at risk of having, having, or who have had, a heart attackor cardiac damage.

Active compounds described herein also include the salts, hydrates,solvates and prodrug forms thereof. The compounds of the presentinvention also include the isomers and metabolites thereof. Certaincompounds of the present invention possess asymmetric carbon atoms(optical centers) or double bonds; the racemates, diastereomers,geometric isomers and individual isomers are all intended to beencompassed within the scope of the present invention. For example, thecompound of the present invention can be the R-isomer or the S-isomer,or a mixture thereof. In addition, the compound of the present inventioncan be the E-isomer or the Z-isomer, or a combination thereof.

Pharmaceutically acceptable salts of the acidic compounds of the presentinvention are salts formed with bases, namely cationic salts such asalkali and alkaline earth metal salts, such as sodium, lithium,potassium, calcium, magnesium, as well as ammonium salts, such asammonium, trimethyl-ammonium, diethylammonium, andtris-(hydroxymethyl)-methyl-ammonium salts. In some embodiments, thepresent invention provides the hydrochloride salt. In other embodiments,the compound is ellipticine hydrochloride.

Similarly acid addition salts, such as of mineral acids, organiccarboxylic and organic sulfonic acids, e.g., hydrochloric acid,methanesulfonic acid, maleic acid, are also possible provided a basicgroup, such as pyridyl, constitutes part of the structure.

The neutral forms of the compounds can be regenerated by contacting thesalt with a base or acid and isolating the parent compound in theconventional manner. The parent form of the compound can differ from thevarious salt forms in certain physical properties, such as solubility inpolar solvents, but otherwise the salts are equivalent to the parentform of the compound for the purposes of the present invention.

The compounds of the present invention can be made by a variety ofmethods known to one of skill in the art (see Comprehensive OrganicTransformations Richard C. Larock, 1989). One of skill in the art willappreciate that other methods of making the compounds are useful in thepresent invention.

Administration of cells or compounds described herein is by any of theroutes normally used for introducing pharmaceuticals. The pharmaceuticalcompositions of the invention may comprise a pharmaceutically acceptablecarrier. Pharmaceutically acceptable carriers are determined in part bythe particular composition being administered, as well as by theparticular method used to administer the composition. Accordingly, thereare a wide variety of suitable formulations of pharmaceuticalcompositions of the present invention (see, e.g., Remington'sPharmaceutical Sciences, 17^(th) ed. 1985)).

Formulations suitable for administration include aqueous and non-aqueoussolutions, isotonic sterile solutions, which can contain antioxidants,buffers, bacteriostats, and solutes that render the formulationisotonic, and aqueous and non-aqueous sterile suspensions that caninclude suspending agents, solubilizers, thickening agents, stabilizers,and preservatives. In the practice of this invention, compositions canbe administered, for example, orally, nasally, topically, intravenously,intraperitoneally, intrathecally or into the eye (e.g., by eye drop orinjection). The formulations of compounds can be presented in unit-doseor multi-dose sealed containers, such as ampoules and vials. Solutionsand suspensions can be prepared from sterile powders, granules, andtablets of the kind previously described. The modulators can also beadministered as part of a prepared food or drug.

The dose administered to a patient, in the context of the presentinvention should be sufficient to induce a beneficial response in thesubject over time, i.e., to ameliorate a condition of the subject. Theoptimal dose level for any patient will depend on a variety of factorsincluding the efficacy of the specific modulator employed, the age, bodyweight, physical activity, and diet of the patient, and on a possiblecombination with other drug. The size of the dose also will bedetermined by the existence, nature, and extent of any adverseside-effects that accompany the administration of a particular compoundor vector in a particular subject. Administration can be accomplishedvia single or divided doses.

VII. Screening for Agents that Induce Pluripotent Stem Cell Development

The present invention provides for methods of screening for agents thatcan “replace” one of the four iPS transcription factors (i.e., an Octpolypeptide, a Klf polypeptide, a Myc polypeptide, and a Soxpolypeptide), or alternatively can replace an Oct polypeptide, a Klfpolypeptide, or a Sox polypeptide in cells where Myc is not necessary toreprogram cells into pluripotent cells (Nakagawa, M. et al. NatureBiotechnol. 26, 101-106 (2007); Wernig, M., Meissner, A., Cassady, J. P.& Jaenisch, R. Cell Stem Cell 2, 10-12 (2008)) or alternatively improvethe efficiency of induction to pluripotency.

In some embodiments, the methods comprise introducing one or moreexpression cassettes for expression of at least one of, but not all of,an Oct polypeptide, a Klf polypeptide, a Myc polypeptide, and a Soxpolypeptide into non-pluripotent cells to generate transfected cells;subsequently contacting the transfected cells to a library of differentagents; screening the contacted cells for pluripotent stem cellcharacteristics; and correlating the development of stem cellcharacteristics with a particular agent from the library, therebyidentifying an agent that stimulates dedifferentiation of cells intopluripotent stem cells. In some embodiments, the cells are contactedwith at least one of an agent that inhibits H3K9 methylation; an L-typeCa channel agonist; an activator of the cAMP pathway; a DNAmethyltransferase (DNMT) inhibitor; a nuclear receptor ligand; a GSK3inhibitor; a MEK inhibitor, a TGFβ receptor/ALK5 inhibitor, a HDACinhibitor; or an Erk inhibitor as well as one or more members of a smallmolecule or other agent library to identify a library member thatinduces or improves induction of cells to pluripotency. Thus, mixturesof non-pluripotent cells and at least one (e.g., 1, 2, 3, 4, 5 or moreof) an agent that inhibits H3K9 methylation; an L-type Ca channelagonist; an activator of the cAMP pathway; a DNA methyltransferase(DNMT) inhibitor; a nuclear receptor ligand; a GSK3 inhibitor; a MEKinhibitor, a TGFβ receptor/ALK5 inhibitor, a HDAC inhibitor; or an Erkinhibitor are provided in the present invention.

The agents in the library can be any small chemical compound, or abiological entity, such as a protein, sugar, nucleic acid or lipid.Typically, test agents will be small chemical molecules and peptides.Essentially any chemical compound can be used as a potential agent inthe assays of the invention, although most often compounds that can bedissolved in aqueous or organic (especially DMSO-based) solutions areused. The assays are designed to screen large chemical libraries byautomating the assay steps and providing compounds from any convenientsource to assays, which are typically run in parallel (e.g., inmicrotiter formats on microtiter plates in robotic assays). It will beappreciated that there are many suppliers of chemical compounds,including Sigma (St. Louis, Mo.), Aldrich (St. Louis, Mo.),Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika(Buchs, Switzerland) and the like.

In some embodiments, high throughput screening methods involve providinga combinatorial chemical or peptide library containing a large number ofpotential iPS replacement agents (potentially acting to replace one ofthe iPS proteins). Such “combinatorial chemical libraries” are thenscreened in one or more assays, as described herein, to identify thoselibrary members (particular chemical species or subclasses) that displaya desired characteristic activity, i.e., such as inducing pluripotentstem cell characteristics in cells that express some, but not all of, anOct polypeptide, a Klf polypeptide, a Myc polypeptide, and a Soxpolypeptide.

A combinatorial chemical library is a collection of diverse chemicalcompounds generated by either chemical synthesis or biologicalsynthesis, by combining a number of chemical “building blocks” such asreagents. For example, a linear combinatorial chemical library such as apolypeptide library is formed by combining a set of chemical buildingblocks (amino acids) in every possible way for a given compound length(i.e., the number of amino acids in a polypeptide compound). Millions ofchemical compounds can be synthesized through such combinatorial mixingof chemical building blocks.

Preparation and screening of combinatorial chemical libraries is wellknown to those of skill in the art. Such combinatorial chemicallibraries include, but are not limited to, peptide libraries (see, e.g.,U.S. Pat. No. 5,010,175, Furka, Int. J. Pept. Prot. Res. 37:487-493(1991) and Houghton et al., Nature 354:84-88 (1991)). Other chemistriesfor generating chemical diversity libraries can also be used. Suchchemistries include, but are not limited to: peptoids (e.g., PCTPublication No. WO 91/19735), encoded peptides (e.g., PCT Publication WO93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091),benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such ashydantoins, benzodiazepines and dipeptides (Hobbs et al., Proc. Nat.Acad. Sci. USA 90:6909-6913 (1993)), vinylogous polypeptides (Hagiharaet al., J. Amer. Chem. Soc. 114:6568 (1992)), nonpeptidalpeptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer.Chem. Soc. 114:9217-9218 (1992)), analogous organic syntheses of smallcompound libraries (Chen et al., J. Amer. Chem. Soc. 116:2661 (1994)),oligocarbamates (Cho et al., Science 261:1303 (1993)), and/or peptidylphosphonates (Campbell et al., J. Org. Chem. 59:658 (1994)), nucleicacid libraries (see Ausubel, Berger and Sambrook, all supra), peptidenucleic acid libraries (see, e.g., U.S. Pat. No. 5,539,083), antibodylibraries (see, e.g., Vaughn et al., Nature Biotechnology, 14(3):309-314(1996) and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang etal., Science, 274:1520-1522 (1996) and U.S. Pat. No. 5,593,853), smallorganic molecule libraries (see, e.g., benzodiazepines, Baum C&EN,January 18, page 33 (1993); isoprenoids, U.S. Pat. No. 5,569,588;thiazolidinones and metathiazanones, U.S. Pat. No. 5,549,974;pyrrolidines, U.S. Pat. Nos. 5,525,735 and 5,519,134; morpholinocompounds, U.S. Pat. No. 5,506,337; benzodiazepines, U.S. Pat. No.5,288,514, and the like).

Devices for the preparation of combinatorial libraries are commerciallyavailable (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, LouisvilleKy., Symphony, Rainin, Woburn, Mass., 433A Applied Biosystems, FosterCity, Calif., 9050 Plus, Millipore, Bedford, Mass.). In addition,numerous combinatorial libraries are themselves commercially available(see, e.g., ComGenex, Princeton, N.J., Tripos, Inc., St. Louis, Mo., 3DPharmaceuticals, Exton, Pa., Martek Biosciences, Columbia, Md., etc.).

Cells contacted with the agents, and optionally expressing some but notall of an Oct polypeptide, a Klf polypeptide, a Myc polypeptide, and aSox polypeptide (e.g., combinations of one, two or three of an Octpolypeptide, a Klf polypeptide, a Myc polypeptide, and a Soxpolypeptide), can then be screened for the development of pluripotentcells, e.g., by screening for one or more pluripotent stem cellcharacteristics. Initial screens can be designed by transforming thecells to be screened with an expression cassette comprising promoterelements known to be activated in pluripotent stem cells (optionally,but not other cells) operably linked to a selectable or otherwiseidentifiable marker. For example, a detectable marker such as GFP orother reporter system can be used. Exemplary promoter elements known tobe activated in pluripotent cells include, but are not limited to, Oct4,Nanog, SSEA1 and ALP promoter sequences. Cells can also be screened forexpression of other pluripotent cell markers (e.g., byimmunofluorescence, etc.) as are known in the art, including, but notlimited to Nanog, SSEA1 and ALP. In some embodiments, cell morphology isexamined.

In some embodiments, the cells are cultured in the presence of aMAPK/ERK kinase (MEK) inhibitor. The inventors have found that thepresence of a MEK inhibitor results in both inhibition of growth ofnon-pluripotent cells and stimulation of growth of pluripotent stemcells. This effect therefore magnifies the “signal” of the screen andallows for more efficient and sensitive detection of agents that inducereprogramming of cells into pluripotent stem cells. A wide variety ofMEK inhibitors are known, including but not limited to, PD0325901 (see,e.g., Thompson, et al., Current Opinion in Pharmacology 5(4): 350-356(2005)); MEK Inhibitor U0126 (Promega), ARRY-886 (AZD6244) (ArrayBiopharma); PD98059 (Cell Signaling Technology); andAmino-thio-acrylonitriles (U.S. Pat. No. 6,703,420). Other MEKinhibitors are described in, e.g., U.S. Pat. No. 6,696,440 and WO04/045617, among others.

VIII. Cell Mixtures

As discussed herein, the present invention provides for non-pluripotentcells in a mixture with one or more compound selected from the groupconsisting of an agent that inhibits H3K9 methylation; an L-type Cachannel agonist; an activator of the cAMP pathway; a DNAmethyltransferase (DNMT) inhibitor; a nuclear receptor ligand; a GSK3inhibitor; a MEK inhibitor, a TGFβ receptor/ALK5 inhibitor, a HDACinhibitor; or an Erk inhibitor. In some embodiments, the compound is inthe mixture at a concentration sufficient to induce or improveefficiency of induction to pluripotency. For example, in someembodiments, the compounds are in a concentration of at least 0.1 nM,e.g., at least 1, 10, 100, 1000, 10000, or 100000 nM, e.g., between 0.1nM and 100000 nM, e.g., between 1 nM and 10000 nM, e.g., between 10 nMand 10000 nM. In some embodiments, the mixtures are in a syntheticvessel (e.g., a test tube, Petri dish, etc.). Thus, in some embodiments,the cells are isolated cells (not part of an animal). In someembodiments, the cells are isolated from an animal (human or non-human),placed into a vessel, contacted with one or more compound as describedherein. The cells can be subsequently cultured and optionally, insertedback into the same or a different animal, optionally after the cellshave been stimulated to become a particular cell type or lineage.

As explained herein, in some embodiments, the cells comprise anexpression cassette for heterologous expression of at least one or moreof an Oct polypeptide, a Myc polypeptide, a Sox polypeptide and a Klfpolypeptide. In some embodiments, the cells do not include an expressioncassette to express any of the Oct, Myc, Sox of Klf polypeptides. Cellswith or without such expression cassettes are useful, for example,screening methods as described herein.

Examples of non-pluripotent cells include those described herein,including but not limited to, cells from a tissue selected from bonemarrow, skin, skeletal muscle, fat tissue and peripheral blood.Exemplary cell types include, but are not limited to, fibroblasts,hepatocytes, myoblasts, neurons, osteoblasts, osteoclasts, and T-cells.

The present invention also provides mixtures (with, or optionallywithout cells) of an agent that inhibits H3K9 methylation (including butnot limited to BIX-01294) with a compound selected from at least one ofan L-type Ca channel agonist; an activator of the cAMP pathway; a DNAmethyltransferase (DNMT) inhibitor; a nuclear receptor ligand; a GSK3inhibitor; a MEK inhibitor, a TGFβ receptor/ALK5 inhibitor, a HDACinhibitor; or an Erk inhibitor. In some including but not limited toembodiments, the agent and at least one compound listed above is at aconcentration as described above. Such mixtures are useful, for example,as “pre-mixes” for induction of pluripotency in cells.

IX. Kits

The present invention also provides kits, e.g., for use in inducing orimproving efficiency of induction of pluripotency in cells. Such kitscan comprise one of more compound selected from the group consisting ofan agent that inhibits H3K9 methylation; an L-type Ca channel agonist;an activator of the cAMP pathway; a DNA methyltransferase (DNMT)inhibitor; a nuclear receptor ligand; a GSK3 inhibitor; a MEK inhibitor,a TGFβ receptor/ALK5 inhibitor, a HDAC inhibitor; or an Erk inhibitor.In some embodiments, the kits comprise an agent that inhibits H3K9methylation (including but not limited to BIX-01294) and a secondcompound (separate or mixed with agent that inhibits H3K9 methylation)selected from at least one of an L-type Ca channel agonist; an activatorof the cAMP pathway; a DNA methyltransferase (DNMT) inhibitor; a nuclearreceptor ligand; a GSK3 inhibitor; a MEK inhibitor, a TGFβ receptor/ALK5inhibitor, a HDAC inhibitor; or an Erk inhibitor.

In some embodiments, the kits further comprise non-pluripotent cells.Examples of non-pluripotent cells include those described herein,including but not limited to, cells from a tissue selected from bonemarrow, skin, skeletal muscle, fat tissue and peripheral blood.Exemplary cell types include, but are not limited to, fibroblasts,hepatocytes, myoblasts, neurons, osteoblasts, osteoclasts, and T-cells.

EXAMPLE Example 1

Toward identifying conditions that can replace viral transduction ofoncogenic transcription factors (e.g. cMyc and Oct4 (Hochedlinger, K. etal., Cell 121, 465-477 (2005)) and enhance reprogramming efficiency, wesought to exploit combination of two approaches: one was to examine adefined progenitor cell type based on the notion that certain accessibleadult progenitor cells may endogenously express at certain level some ofthe required genes for inducing pluripotency and/or the loci of thesegenes may be less silenced so that such progenitor cells might be moreefficiently reprogrammed and/or with less genetic manipulations; theother approach was to screen small molecules that may be able to replaceviral integration of specific transcription factor and/or promotereprogramming process.

Among various adult stem/progenitor cells that are accessible fromdifferent tissues, we initially focused our efforts on neural progenitorcells for the following reasons: (i) In contrast to heterogeneousprimary fibroblast culture (e.g., MEF) which may contain various typesof stem/progenitor cells, neural progenitor cells are relatively definedpopulation of cells and can be clonally expanded under chemicallydefined conditions. (ii) Neural progenitor cells endogenously expressspecific Sox genes (e.g. Sox1 or Sox2), which, although is at lowerlevel than overexpression, might be sufficient for generating iPS cells.(iii) Neural progenitor cells or Sox gene expressing cells may beisolated from other tissues (Fernandes, K. J. L. et al., Nature CellBiology 6, 1082-1093 (2004); Seaberg, R. M. et al., Nature Biotechnol.22, 1115-1124 (2004)) and expanded in vitro. Therefore, defined neuralprogenitor cells represent an excellent model system to address abovequestions in reprogramming process/mechanism. To establish an unlimited,highly reproducible and defined source of neural progenitor cells thatcan be used in high throughput screens, we chose to use mESC-derivedneural progenitor cells that contain a GFP-IRES-Puro/GiP reporter undercontrol of the Oct4 regulatory elements, since mESCs can be grown inlarge quantity and their differentiation to a homogenous population ofneural progenitor cells is well defined (Conti, L. et al., PLoS Biol. 3,e283 (2005)), as well as the validated reporter activity (Ying, Q. L. etal., Nature 416, 545-548 (2002)) can facilitate facile assay detection.

The reporter neural progenitor cells were generated using a wellestablished protocol by differentiating the Oct4-GiP mESCs grown inmonolayer on gelatin in a chemically defined medium/CDM condition in theabsence of serum and other growth factors/cytokines at low cell densityfor eight days, followed by neurophere formation and subsequent serialpassaging in single cells in neural cell expansion media supplementedwith 10 ng/ml of bFGF and EGF in monolayer for over six passages/24days. The resulting neural progenitor cells were homogenous by cellmorphology and neural marker expression, and were confirmed to be GFPnegative and puromycin sensitive. Such neural progenitor cells plated inmonolayer in conventional mESC growth media were transduced withcombinations of four, three, or two of the four factors, followed bytreating the transduced cells with individual small molecules from asmall known drug collection in a typical 6-well format. The compoundtreatment and culture were continued for additional ten days beforepuromycin was added. The number of green and puro-resistant colonies wascounted at day 14. In comparison to the only four-gene transduced neuralcells as the positive control, compound conditions that generated moregreen colonies than the corresponding gene-only conditions were pickedas primary hits. To further confirm these primary hit conditions, wechose to use a late passage of mouse CNS neural progenitor cells (Do, J.T. et al., Stem Cells 25, 1013-1020 (2007)) that were derived from fetalbrain of OG2^(+/−)/ROSA26^(+/−) transgenic mice (which contain anOct4-GFP reporter) and expanded in monolayer under the same neural CDMcondition as the above with 10 ng/ml of bFGF and EGF. Such cells trulyfrom a non-pluripotent tissue would be devoid of concerns of anycontaminating ES cell in the above screening system, which is althoughhighly unlikely with all appropriate controls. Similar cultureconditions and reprogramming assays were performed using the OG2 neuralprogenitor cells except that puromycin was not used and green colonieswere picked out and characterized by staining of Nanog, SSEA1 and ALP.We found that almost all of the green colonies that can be identified atday 12-14 can be expanded to long-term stable iPS cells that areindistinguishable from the classic mESCs by morphology and typicalpluripotency marker expression.

We first focused our characterization efforts on two new conditions thatcould be reconfirmed using the fetal neural progenitor cells. Just ashypothesized that certain tissue-specific progenitor cells withendogenous expression of certain relevant reprogramming genes mayrequire less exogenous genetic manipulation to generate iPS cells, wefound that viral transduction of only Oct4 and Klf4 together issufficient to generate iPS cells from neural progenitor cells in 10-14days. While such reprogramming efficiency (1-2 GFP colonies per 3.5×10⁴cells) is lower than conditions with additional Sox2 and cMyc viraltransduction (8-10 GFP colonies per 3.5×10⁴ cells) (FIG. 1), it isinteresting that the reprogramming kinetics by the two genes only (Oct4and Klf4) is not significantly slower than that by the original fourgenes. This is in contrast to the recent observations that omitting cMycin generating iPS cells from MEFs is significantly slower (e.g.additional 2 weeks) than the condition having cMyc overexpression eventhe embryonic fibroblasts endogenously express cMyc. Most interestingly,we found that a small molecule, BIX01294 (Kubicek, S. et al., MolecularCell 25, 473-481 (2007)) that specifically inhibits G9a (a histonemethyltransferase for H3K9me2), can significantly improve thereprogramming efficiency to or higher than the level of using viraltransduction of all four factors, while it didn't significantly shortenthe kinetics of reprogramming. The reprogramming event is typicallyassayed by the ability of identifying the iPS cell colonies, which isinfluenced by many factors, including the methods of cell culture, cellidentification and/or selection, as well as the input cell type, number,and reprogramming efficiency and kinetics. Consequently, a requirementfor any given gene for reprogramming is relative to that specificsetting and largely depends on the reprogramming efficiency/kinetics. Inthis regard, this single small molecule, BIX01294, functionally replacedviral transduction of cMyc and Sox2 to large extent.

GFP+ iPS cell colonies readily appeared 12 days after OG2 neuralprogenitor cells were transduced with Oct4/Klf4 retroviruses and treatedwith BIX01294. Day 14 iPS cells generated from Oct4-Klf4 viraltransduction and BIX01294 treatment can be readily expanded in theconventional mESC culture condition on MEF feeder cells in the presenceof LIF without the requirement of continued BIX01294 treatment. iPScells generated by Oct4/Klf4 viral transduction and BIX01294 treatmentcan long-term self-renew on MEF feeders in the mESC growth media withoutcontinued BIX01294 treatment. They grow as compact and domed colonies.These iPS cells maintain characteristic mESC-colony morphology,homogenously express typical pluripotency markers in comparable level asmESCs, including Oct4, Nanog, SSEA1 and ALP by immunocytochemistry,histostaining and RT-PCR analysis. Furthermore, such iPS cells, whichhad been serially passaged over 10 passages, can effectivelydifferentiate into characteristic neurons (βIII-tubulin), beatingcardiomyocytes (cardiac troponin) and pancreatic or hepatic cells (Pdx1or Albumin), derivatives of the three primary germ layers under standardembryoid body or directed differentiation methods. And most importantly,such iPS cells can efficiently incorporate into the ICM of a blastocystafter aggregation with an 8-cell embryo, lead to a high-grade chimerismafter the aggregated embryos were transplanted into mice, and contributeto the germ line in vivo. These in vitro and in vivo characterizationsconfirm that the iPS cells generated by Oct4 and Klf4 viral transductionwith simultaneous BIX01294 treatment are morphologically, functionallyand by typical pluripotency marker expression indistinguishable from theoriginal four-factor iPS cells and the classic mESCs.

One question is whether expression of Oct4, Sox2, Klf4 and cMyc,regardless of endogenously or exogenously, would be a prerequisite forgenerating iPS cells. Interestingly, recent reprogramming studies ongenerating human iPS cells from fibroblasts have shown while exogenousexpression of Klf4 and cMyc are functionally exchangeable with Nanog andLin28, expression of Oct4 and Sox2 seems to be required so far from allpublished iPS cell studies. Interestingly, we found that viraltransduction of Klf4, Sox2, and cMyc with simultaneous BIX01294treatment in the absence of Oct4 expression can also generate iPS cells(FIG. 2) while viral transduction of such three factors/KSM alone failedto produce any iPS cell colony under our assay conditions. Similarly,such KSM-BIX01294 generated iPS cells can be stably expanded andlong-term self-renew on MEF feeders in the conventional mESC growthconditions over passages without BIX01294, maintain the characteristicmESC morphologies, homogenously express typical pluripotency markers,including ALP, Oct4, Nanog and SSEA1, and differentiate into cells inthe three germ layers in vitro. It should be noted that thereprogramming efficiency in the absence of Oct4 expression is relativelylow.

Finally, we observed that the application of a specific small moleculeinhibitor of MEK, PD0325901, to late stage of reprogramming (e.g. afterOct4-GFP activation) can serve as an excellent selection strategy forgenerating iPS cells. Due to the very low efficiency of reprogramming,iPS cells are typically selected out by utilizing reporter (e.g.Neo/Puro or GFP) that is under control of the regulatory elements of apluripotency marker using genetically modified somatic cell lines, ormanually picked out based on cell morphology. While the later methodapplicable to genetically unmodified cells is better suited for ultimateclinical application of iPS cells, it is a much more tedious and lessreliable technique that typically requires picking and propagating overseveral passages many colonies, only a small fraction of which wouldbecome true iPS cells efficiently. This is partly because that largepercentage of similarly looking colonies may be partially reprogrammedcells and/or simply transformed cells, which grow rapidly and mayinterfere with growth and reprogramming of iPS cells. Consequently,having an alternative selection strategy for genetically unmodifiedcells would be highly desirable. We found that PD0325901 inhibits growthof non-iPS cells while it effectively promotes growth and stablereprogramming of iPS cells, leading to larger and more homogenouscolonies of iPS cells. This observation might be partly due to themechanism that MEK activity is required for cell cycle progression ofsomatic cells, while mESCs lack of such restriction for growth andinhibition of MEK also inhibits differentiation of mESCs (contributingto further stabilization of the iPS cell state).

The results presented here have a number of important implications. (1)The lower endogenous expression level (than overexpression) of criticalgenes required for reprogramming by (tissue-specific progenitor) somaticcells may be sufficient to replace the corresponding exogenous geneexpression via viral transduction for generating iPS cells. This pointsto an alternative strategy of generating iPS cells from somatic cellsusing less genetic manipulation by exploiting practically accessiblecells that endogenously express certain relevant reprogramming genes viaan intrinsic tissue specificity and/or ex vivo culture manipulation. (2)This is a proof-of-principle demonstration that small molecules can beidentified from rationally designed cell-based screens to functionallyreplace viral transduction of certain transcription factor(s), improvereprogramming efficiency, or serve as a selection condition ingenerating iPS cells. Not only may such pharmacological approach forreplacing specific genetic manipulation substantially reduce risksassociated with insertion of oncogenes (e.g. cMyc and Oct4) andinsertional mutagenesis, but also it opens up the possibility ofenabling a precisely controlled and highly efficient reprogrammingprocess by defined small molecules. This is especially important forstudying the molecular mechanism of reprogramming, which currently islargely intractable due to its very low efficiency and slow kinetics.(3) In contract to the gain-of-function approach in generating iPScells, the highly effective use of those specific small moleculeinhibitors suggests that loss-of-function of specific genes may be atleast equally important and effective in generating iPS cells. Moreimportantly, the function of BIX01294 defines a specific epigeneticmechanism/target, i.e. inhibition of G9a-mediated H3K9me2, in generatingiPS cells. This is consistent with the previous findings that therepressive H3K9 methylation is associated with Oct4 inactivation duringdifferentiation (Feldman, N. et al., Nature Cell Biology 8, 188-194(2006)), and histone lysine methylation, although robust, is dynamic andregulated by HMTases and lysine demethylases. BIX01294 may function tofacilitate shifting the epigenetic balance from a silenced state of Oct4to an active transcription. (4) Exemplified by using MEK inhibitor forfacile selection of iPS cells, exploiting the differences betweensomatic cells and ESCs by small molecules represents analternative/attractive strategy for selecting iPS cells. Finally, it isconceivable that the strategies and small molecules reported here can befurther explored for improved approaches and better mechanisticunderstanding of generating iPS cells, and combined with additionalsmall molecules (that can replace the function of remaining transducedtranscription factors and improve reprogramming) as well as othernon-genetic methods (e.g. protein transduction) to ultimately allowgeneration of iPS cells in high efficiency in a completely chemicallydefined condition without any genetic modification.

Methods.

Neural progenitor cell culture. Neural progenitor cells were derivedfrom mESCs or mouse fetal brains according to the protocol reported byConti et al (Conti, L. et al., PLoS Biol. 3, e283 (2005)). Briefly,mESCs were plated onto a 0.1% gelatin-coated dish at 1×10⁴ cells/cm² inthe neural induction medium (50% DMEM/F12 basal medium, 50% Neurobasalmedium, 0.5×N2, 0.5×B27, 1× Glutamax, 50 ug/ml BSA) and differentiatedfor 7-8 days. The formed neural rosettes were then trypsinized intosingle cells and replated into an Ultra-Low Attachment dish (Corning) toform neurosphere in the neural progenitor cell expansion medium(DMEM/F12, 1×N2, 10 ng/ml bFGF, 10 ng/ml EGF, 50 ug/ml BSA). After threedays in suspension neurospheres were re-attached to a gelatin-coateddish and further differentiated for 4-6 days before they were furtherpassaged in single cells and grown in monolayer on gelatin-coated dishesin the neural progenitor cell expansion medium for over 5-6 passages.

Neurospheres from brains of 12.5 to 16.5 dpc ROSA26/OG2 heterozygousfetuses were generated as previously described (Do, J. T. et al., StemCells 25, 1013-1020 (2007)). Briefly, the cortex was dissected,enzymatically dissociated, and passed through a 70 μm nylon mesh(Falcon; Franklin Lakes, N.J.). Neural cells were further purified bycentrifugation in 0.9 M sucrose in 0.5×HBSS at 750 g for 10 min and in4% BSA in EBSS solution at 200 g for 7 min. Such cells were furthergrown in suspension to form neurospheres and subsequently seriallypassaged in monolayer on gelatin-coated dishes in the neural progenitorcell expansion medium as described above. Animal experiments wereapproved and performed according to the Animal Protection Guidelines ofthe Government of Max Planck Society, Germany.

Retrovirus Transduction.

The murine cDNAs for Oct4, Klf4, Sox2 and c-Myc were cloned into thepMSCV retroviral vector and verified by sequencing. The pMX-basedretroviral vectors were obtained from Addgene. The virus production andtransduction were performed as described 2-3.

iPS Cell Induction from Neural Progenitor Cells.

mESC-derived or primary OG2 mouse neural progenitor cells were platedinto Matrigel (1:50, BD Biosciences) coated 6-well plates at 3.5×10⁴cells/well in the neural progenitor cell expansion media. After one daythese cells were transduced with retrovirus for overnight, and themedium was changed into the mESC growth media [DMEM, 5% FBS, 10% KSR, 1×non-essential amino acids (Gibco), 2 mM L-glutamine (Gibco), 0.1 mMβ-mercaptoethanol (Gibco) and 10³ unit/ml LIF (Chemicon)] with orwithout BIX01294 (0.5-1 μM). GFP-positive iPS cell colonies appearedafter 9-14 days, and were picked out and expanded on MEF feeder cellswith the mESC growth media.

Characterization Assays.

ALP staining was performed as instructed by the Alkaline PhosphataseDetection Kit (Chemicon). Cells were fixed in 4% paraformaldehyde,washed three times by PBS, and then incubated in PBS containing 0.3%TritonX-100 (Sigma) and 10% normal donkey serum (Jackson ImmunoResearch)for 30 min at room temperature. The cells were then incubated withprimary antibody overnight at 4° C.: mouse anti-Oct4 antibody, mouseanti-SSEA1 antibody (1:200, Santa Cruz), rabbit anti-Sox2 antibody(1:200, Chemicon), rabbit anti-Nanog antibody (AbCam), rabbit anti-Pdx1(1:200, from Dr. C. Wright), mouse anti-βIII-tubulin antibody (1:500,Covance), mouse anti-cardiac troponin T (1:200, DSHB), rabbitanti-Albumin antibody (DAKO). After washing, cells were furtherincubated with secondary antibodies: Alexa Fluoro555 donkey anti-mouseIgG or Alexa Fluoro555 donkey anti-rabbit IgG (1:500, Invitrogen) for 30min at RT. Nuclei were detected by DAPI (Sigma) staining. Images werecaptured by Nikon TE2000-U.

Aggregation of iPS Cells with Zona-Free Embryos.

iPS cells were aggregated with denuded post-compacted eight-cell stageembryos to obtain aggregate chimera. Eight-cell embryos (B6C3F1) flushedfrom females at 2.5 dpc were cultured in microdrops of KSOM medium (10%FCS) under mineral oil. Clumps of iPS cells (10-20 cells) after shorttreatment of trypsin were chosen and transferred into microdropscontaining zona-free eight-cell embryos. Eight-cell embryos aggregatedwith iPS cells were cultured overnight at 37° C., 5% CO2. Aggregatedblastocysts developed from eight-cell stage were transferred into oneuterine horn of a 2.5 dpc pseudopregnant recipient.

Example 2

Somatic cells can be induced into pluripotent stem cells (iPSC) with acombination of four transcription factors, Oct4/Sox2/Klf4/c-Myc orOct4/Sox2/Nanog/LIN28. This provides an enabling platform to obtainpatient specific cells for various therapeutic and researchapplications. However, several problems remain for this approach to betherapeutically relevant due to drawbacks associated with efficiency andviral genome-integration. As explained above, neural progenitor cells(NPCs) transduced with Oct4/Klf4 can be reprogrammed into iPSCs.However, NPCs express Sox2 endogenously, possibly facilitatingreprogramming in the absence of exogenous Sox2. In this study, weidentified a small molecule combination, BIX-01294 and BayK8644, thatenables reprogramming of Oct4/Klf4 transduced mouse embryonicfibroblasts, which do not endogenously express the factors essential forreprogramming. This study demonstrates that small molecules identifiedthrough a phenotypic screen can compensate for viral transduction ofcritical factors, such as Sox2, and improve reprogramming efficiency.

This example is aimed to assess if small molecules can replace specificviral transduction to obtain iPSCs from a general cell lineage, in whichnone of the TFs deemed essential for reprogramming, Oct4, Sox2 and Klf4,are expressed. Hence, mouse embryonic fibroblasts (MEFs) were used.Finding a small molecule that could replace one of these TFs in theinduction of MEF reprogramming might lead to the identification ofgeneral pathways involved in this process. Such chemical strategy mightbe more amenable for therapeutic application. Consequently, we screeneda collection of known drugs to identify small molecules that can enablethe generation of iPSCs from MEFs transduced with OK, and thus, couldcompensate for the lack of Sox2 overexpression. Through the differentscreens performed we identified that a combination of BIX with Bayk8644(BayK), a L-channel calcium agonist (Schramm, M. et al., Nature,303:535-537 (1983)) was one of the most effective. Bayk was of interestbecause it exerts its effect upstream in cell signaling pathways, anddoes not directly cause epigenetic modifications. It is likely that thistype of molecule, such as BayK or activators of the Wnt signalingpathway (Marson, A. et al., Cell Stem Cell, 3:132-135 (2008)), can beexploited to induce reprogramming in a more specific manner thanmolecules acting directly at the epigenetic level causing DNA or histonemodification. Some of these epigenetic modifiers have already been shownto facilitate the reprogramming process, such as BIX (Shi, Y. et al.,Cell Stem Cell, 2:525-528 (2008)), valproic acid (Huangfu, D. et al.,Nat Biotechnol, 26:795-797 (2008)) and 5′ azacytidine (Mikkelsen, T. etal., Nature, 454:49-55 (2008)).

This present study demonstrates that small molecules identified througha phenotypic screen can be used to effectively compensate for the viraltransduction of another critical iPSC TF, Sox2, which is notendogenously expressed in fibroblasts. Moreover, it highlights theimportant contribution that small molecule screens will eventually maketo the discovery of new molecular targets and mechanisms involved incomplicated biological processes such as reprogramming.

Results

Phenotypic Screen Leads to the Discovery of Small Molecules that EnableMEF Reprogramming when Transduced with Only Two TFs.

Unmodified MEFs derived from E13-14 embryos of the 129 mice were usedfor the initial screen. MEFs were plated on Matrigel at 3.5×10⁴cells/well of a 6-well plate and transduced with OK (retroviral vectorsexpressing Oct4 and Klf4) alone. Within 14-21 days, treated cells wereassessed for the appearance of colonies that had the characteristicembryonic stem cell (ESC), colony morphology and were positive for thepluripotency marker alkaline phosphatase (ALP). Such OK-transduced cellsgenerated only a few small non-compact colonies, which were weaklypositive for ALP expression. These colonies initially appeared within 21days after viral transduction and were difficult to expand. Therefore,such assay system offered a clean background for the identification ofsmall molecules having desirable reprogramming inducing activity. Usingthis system, compounds from a library of around 2000 known smallmolecules (see Experimental Procedures below) were screened and wereidentified as hits when they induced the appearance of ESC colonies thatwere strongly positive for ALP within 14-21 days after treatment. Thisimage-based method provided a more accurate assessment of reprogrammingas compared to homogenous reporter-based assay. BIX appeared to have thestrongest effect with reproducible induction of more than 1-2 compactESC-like colonies with high ALP expression. We observed that when MEFswere treated with BIX after OK viral transduction, compact colonies withstrong ALP expression could be readily detected within approximately14-21 days. These cells were also positive for Nanog, Oct4 and SSEA-1expression. This result, obtained with a more general cell type, whichdoes not endogenously express any of the three essential reprogramminggenes, further validates our previous observation that BIX has strongreprogramming inducing activity and inhibition of the G9a HMTase canfacilitate reprogramming (Shi, Y. et al., Cell Stem Cell, 2:525-528(2008)). However, the reprogramming efficiency in MEFs transduced withOK and treated with BIX was still low, about 2 colonies/3.5×10⁴ cells,in comparison to the four factor-induced reprogramming of MEFs or theOK/BIX NPC reprogramming (Shi, Y. et al., Cell Stem Cell, 2:525-528(2008)). Therefore, we conducted a second screen using a similarprotocol, but where BIX was added to the basal media after OK viraltransduction. This provided a more permissive platform to identify newsmall molecules that could further improve reprogramming efficiency.More importantly, this second screen could facilitate discovery of smallmolecules that impact reprogramming in a more specific manner, forexample by acting on signal transduction pathways rather than on histoneor DNA modifying enzymes. In this second screen, we again assayed thelibrary of around 2000 known small molecules (see ExperimentalProcedures), and confirmed two compounds that were able to act in asynergistic manner with BIX to improve reprogramming based on thecriteria of the screen. One example is RG108, a DNA methyltransferase(DMNT) inhibitor (Brueckner, B. et al., Cancer Res, 65:6305-6311(2005)), which enhanced reprogramming of OK transduced MEFs in thepresence of BIX (FIG. 3). However, similarly to BIX, RG108 is known toimpact the cells at a general epigenetic level, and another DNAmethyltransferase inhibitor, 5-azacytidine has already been shown toenhance reprogramming (Mikkelsen, T. S. et al., Nature, 454:49-55(2008)). Therefore, RG108 was not pursued further for this study.Instead, we focused our phenotypic and functional characterization onanother small molecule that was identified in the second screen, BayK,an L-calcium channel agonist. This small molecule, which showed thestrongest effect in the screen aside from known DNA/histone modifiers,was studied further because it has no observable reprogramming activityon OK-transduced MEFs in the absence of BIX and is not known to impactthe cells directly at the epigenetic level, but rather at the cellsignal transduction level. Therefore, BayK might play a more specificrole in the reprogramming process. When 129 MEFs were transduced withempty retrovirus (negative control); no colonies observed. When 129 MEFswere transduced with OK without small molecules; few small flattenedcolonies with weak ALP expression present. ESC-like iPSC colonies wereobserved 14-21 days after 129 MEFs were transduced with OK and treatedwith BIX/BayK; these ESC-like colonies exhibited strong ALP expression.When OK-transduced MEFs were treated with BIX in combination with BayK,a significant increase in the number of ALP⁺ colonies that closelyresemble the mESC morphology could be observed (˜7 colonies) as comparedto OK-transduced MEFs treated with BIX alone (˜2 colonies). Furthercharacterization of these primary iPSC colonies showed that they werepositive for typical pluripotency markers such as Oct4, Sox2, Nanog, andSSEA1 as determined by immunofluorescence.

iPSCs Obtained from MEFs Transduced with OK and Treated with BIX/BayKhave Pluripotency Properties Characteristic of mESCs.

To further confirm and characterize that OK transduction and BIX/BayKtreatment can induce MEFs to become iPSCs, we used primary MEFs derivedfrom OG2^(+/−)/ROSA26^(+/−) (OG2) transgenic mice, which contain anOct4-GFP reporter (Do, J. T. and Scholer, H. R., Stem Cells, 22:941-949(2004)). Once reprogrammed, these cells could then be used toconveniently assess chimera and germline competency. Similarly to 129MEFs, OG2 MEFs transduced with OK could generate iPSCs when treated witha combination of BayK/BIX (OK2B iPSCs) (FIG. 3). GFP⁺ iPSC coloniescould be first detected on day 14-21 after viral transduction andcompound treatment. When OG2 MEFs were transduced with OK and nottreated with any compounds, only a few small colonies appeared for anaverage of 0.5±0.7 colony per 3.5×10⁴ cells. These colonies weredifficult to passage and therefore were not studied any further.Treatment of OK transduced OG2 MEFs with BIX alone readily andreproducibly enabled reprogramming as compared to OK alone, with 2.5±0.7colonies per 3.5×10⁴ cells. There was a further significant improvementin the reprogramming efficiency when OG2 MEFs transduced with OK weretreated with the combination of BIX (2 μM) and BayK (2 μM), where weobserved 7.7±1.5 colonies per 3.5×10⁴ cells (FIG. 3). Treatment ofOK-transduced OG2 MEFs with BayK alone, in the absence of BIX, did notincrease reprogramming efficiency above OK-transduced MEF control (datanot shown).

OK2B colonies were picked out and serially expanded on irradiated MEFfeeder cells in the conventional mESC growth conditions in the absenceof small molecules for more than 20 passages. Staining and/or RT-PCR(FIG. 4A) showed that these GFP⁺ OK2B iPSCs express typical pluripotencymarkers, including ALP, Nanog, Sox2, Oct4, SSEA1, c-Myc, eRas, Esg1,Ecat1, and Fgf4. RT-PCR assay also demonstrated that OK2B iPSCsexpressed endogenous Oct4 and Klf4 (FIG. 4A). Bisulphite genomicsequencing analyses of the Nanog promoter revealed that it isdemethylated in OK2B iPSCs similarly to the mESC control (R1), while theMEFs' Nanog promoter was hypermethylated (FIG. 4B). This result furthersuggests a reactivation of the stem cell transcription program in theseOK2B iPSCs. In addition, transcriptome analysis showed that expressionprofile of OK2B iPSCs is greatly similar to the one of mESCs with aPearson correlation value of 0.96, while significantly different toMEFs' profile with a Pearson correlation value of 0.84 as exemplified inthe clustering analysis.

For comparison of OK2B transcriptome with mES cells and MEF cells,transcriptome analysis was carried out. RNA was extracted from OK2B iPScells at passage 13 using Qiagen RNAeasy Mini Kit. RNA expression datafor OK2B iPSCs was generated from polyA RNA using GeneChip Mouse Genome430 2.0 Arrays (Affymetrix). Expression data for MEF cells and mES cellswere obtained from the Gene Expression Omnibus (GEO) websitehttp://www.ncbi.nlm.nih.gov/geo/. mES cells data registry number:GSM198062. GSM198063, and GSM198064. MEF cell data registry number:GSM198070 and GSM198072. Pre-processing, normalization (GC-RMA) andhierarchical clustering were performed using dChip(http://biosun1.harvard.edu/complab/dchip/; (Distance metric:correlation (Pearson); linkage method: centroid; gene ordering: bycluster tightness). p value for OK2B iPSC versus MEF cells: 0.84; OK2BiPSC versus mES cells: 0.96. p values were obtained using a Pearsoncorrelation test.

OK2B iPSCs Differentiate into Cells from all Three Germ Layers andContribute to Germline Transmission.

OK2B iPSCs could efficiently form embryoid bodies (EB) in suspension,which could differentiate into endodermal cells (Albumin and Pdx1),mesodermal cells/cardiac muscle cells (CT3) and ectodermal cells/neurons(βIII-tubulin, Tuj1), derivatives of the three primary germ layers. Inaddition, OK2B iPSCs could efficiently incorporate into the inner cellmass of a blastocysts following aggregation with an 8-cell embryo, andlead to chimerism with germline contribution in vivo after theaggregated embryos were transplanted into pseudo-pregnant mice.Moreover, mating of one adult male progeny obtained from theseblastocysts with a female CD1 wild-type mouse led to the production ofLacZ⁺ progeny, three of which showed Oct4-GFP⁺ gonads further validatingthat these iPSCs could contribute to germline transmission. These invitro and in vivo characterizations confirm retroviral transduction withonly two genes, OK, and in conjunction with BIX/BayK treatment aresufficient to reprogram MEFs into iPSCs, which are phenotypically andfunctionally similar to the classic mESCs.

Discussion

The studies presented here provide a proof-of-principle demonstrationthat small molecules can be identified from rationally designedphenotypic screens to functionally replace viral transduction of certainTF(s) as well as improve reprogramming efficiency in generating iPSCsfrom a general cell-type, like MEFs. Such a chemical approach for thegeneration of iPSCs, which offers more precise and temporal control ofthe target/process, would be advantageous over the genetic manipulationwith oncogenes that may also introduce harmful hard-to-detectinsertional genomic alterations. Similar strategies are being used tofind additional small molecules that may ultimately allow reprogrammingof lineage-restricted cells to pluripotent or multipotent state in acompletely chemically defined condition. BIX was originally identifiedand characterized as a specific inhibitor for G9a HMTase (Kubicek, S. etal., Mol Cell, 25:473-481 (2007)). It has been shown to reduce H3K9me2levels at G9a target genes (Feldman, N. et al., Nat Cell Biol, 8:188-194(2006)). Interestingly, histone H3K9 methylation, mediated by G9a, andheterochromatinization represent a highly specific mechanism forepigenetic silencing of embryonic genes such as Oct4 and Rex1 (Feldman,N. et al., Nat Cell Biol, 8:188-194 (2006)). Furthermore, it was alsodemonstrated that knock-down of G9a can assist in fusion-basedreprogramming of adult neuronal cells (Ma, D. K. et al., Stem Cells,26:2131-2141 (2008)). It is therefore fitting that we previouslyobserved that BIX can facilitate the generation of iPSCs from NPCstransduced with either OK or Klf4/Sox2/c-Myc (Shi, Y. et al., Cell StemCell, 2:525-528 (2008)), suggesting that it can compensate for theexogenous expression of Sox2 or Oct4. However, NPCs already expresssignificant levels of Sox2, which might cause these cells to be moresusceptible to reprogramming in the conditions mentioned above. Thispresent study aimed at identifying small molecules that can enablereprogramming of MEFs, which do not express any of the TFs deemednecessary for reprogramming. It was fortuitous that we identified BIX inboth the NPC and MEF screens, and further confirm this molecule has arole in enabling and improving the generation of iPSCs from somaticcells. Given BIX's characterized mechanism of action, our studiespotentially identified a molecular target whose loss-of-function viapharmacological inhibition is sufficient to compensate for thegain-of-function of an essential iPSC reprogramming gene. It furthermechanistically links a specific epigenetic process, inhibition ofG9a-mediated H3K9me2, to iPSC generation. BIX may function to facilitateshifting of the epigenetic balance from a silenced state of pluripotencygenes to an active transcription state. Obviously, combination of BIXwith other chromatin-modifying small molecules, which have differenttargets and mechanisms of action, such as RG108 could be exploited forbetter reprogramming. On the other hand, our observation that BayK, witha characterized activity as a specific L-type calcium channel agonist(Schramm, M. et al., Nature, 303:535-537 (1983)), improves reprogrammingefficiency is intriguing. L-type calcium channels are known to mediateintracellular processes in different tissues such as blood pressureregulation, smooth muscle contractility, insulin secretion, cardiacdevelopment, etc (Tosti, E., Reprod Biol Endocrinol, 4:26 (2006)).Furthermore, activation of L-type calcium channels by differentagonists, including BayK, has been shown to induce intracellularsignaling through CREB activation, sarcoplasmic reticulum Ca²⁺ release,and change in cAMP activity. More importantly, some reports suggest thatcalcium might play a role in the control of mES cell proliferation (Heo,J. S. et al., Am J Physiol Cell Physiol, 290:C123-133 (2006)). However,in our hands, treatment of mES cell with 2 μM BayK alone or incombination with 1 μM BIX does not lead to a change in proliferation(FIG. 5). Furthermore, treatment of OG2 MEF with 2 μM BayK alone or incombination with 1 μM BIX does not induce SOX2 expression (FIG. 6).Needless to say, more work needs to be performed to dissect the precisemechanism by which BayK impacts the reprogramming process. However, itis interesting to find that a small molecule with activity in signalingpathways that have not been previously linked to reprogramming cansignificantly enhance its efficiency. So far, it is the first smallmolecule of its type, aside from Wnt3 protein (Marson, A. et al., CellStem Cell, 3:132-135 (2008)), to show an effect on reprogramming withoutacting directly on chromatin modifiers. As, up to date, most of theother small molecules found to impact reprogramming appear to directlymodify the epigenetic status of the cell: i.e., BIX (Shi, Y. et al.,Cell Stem Cell, 2:525-528 (2008)), valproic acid (Huangfu, D. et al.,Nat Biotechnol, 26:795-797 (2008)) and 5′ azacytidine (Mikkelsen, T. S.et al., Nature, 454:49-55 (2008)). Importantly, BayK seems to haveseveral important characteristics that would be ultimately desirable fora molecule to be therapeutically relevant for in vivo reprogrammingand/or regeneration. The fact that it does not act/reprogram on its own,but needs the presence of BIX to exert its effects suggests that cellsthat are already undergoing a form of reprogramming, perhaps caused byinjury, might be more susceptible to its effect. This might allow us toultimately reprogram the target cell in a more specific manner, withoutimpacting healthy cells systemically, as direct epigenetic modifiersmight.

In summary, we have identified defined small molecule conditions, i.e.,BIX, and combinations of BIX/BayK, or BIX/RG108, which can enable andimprove reprogramming of fibroblasts into iPSCs in conjunction with thetransduction of only two TFs: Oct4 and Klf4. This study further confirmsthe usefulness of a phenotypic screening approach in identifying smallmolecules that can effectively compensate for the viral transduction ofan essential iPSC TF, such as Sox2 in this study or Oct4 as previouslyreported (Shi, Y. et al., Cell Stem Cell, 2:525-528 (2008)). Ultimately,phenotypic small molecule screens may lead to the identification ofsmall molecule that will become powerful tools in providing us with newinsights into the reprogramming process, and may ultimately be useful toin vivo stem cell biology and therapy.

Experimental Procedures

MEFs Derivation

129S2/SvPasCrlf or ROSA26^(+/−)/OG2^(+/−) MEFs were derived according tothe protocol reported on WiCell Research Institute website:“Introduction to human embryonic stem cell culture methods.” Animalexperiments were performed according to the Animal Protection Guidelinesof the Max Planck Institute for Biomolecular Research, Germany.

Retrovirus Transduction and Compounds

pMX-based retroviral vectors for mouse Oct4, Klf4, c-Myc and Sox2 wereobtained from Addgene (Cambridge, Mass.). The viral production andtransduction process was performed as described (Takahashi, K. et al.,Cell, 131:861-872 (2007)). The synthesis and full characterization ofcompound BIX-01294 was as previously described (Kubicek, S. et al., MolCell, 25:473-481 (2007)) and Bayk8644 was purchased from EMD/CalbiochemBiochemical (San Diego, Calif.).

Screen for iPSC Generation from MEFs

For the primary and secondary screens, a collection of known compoundswas used. This collection was composed of roughly 2000 known bioactivemolecules that are commercially available, including FDA-approved drugs,known inhibitors and activators of characterized enzymes (includingLOPAC collection from Sigma-Aldrich (St. Louis, Mo.), Known BioactiveLibrary from BIOMOL (Plymouth Meeting, Pa.) and non-overlapping knowncompounds from EMD Calbiochem (San Diego, Calif.)).

Primary 129S2/SvPasCrlf (primary screen) or ROSA26^(+/−)/OG2^(+/−)(secondary screen) MEFs were plated onto Matrigel (1:50; BD Biosciences,Bedford, Mass.) coated dishes at a density of 3.5×10⁴ cells per well ofa 6-well plate. Twenty-four hours later, these cells were transducedovernight with defined retroviruses at 37° C., 5% CO₂. Twelve tofourteen hours later, the media on the transduced cells was changed tomESC medium [Knockout DMEM, 10% ES-qualified FBS, 10% Knockout serumreplacement, 1% Glutamax, 1% Non-essential amino acids,penicillin/streptomycin, 0.1 mM β-mercaptoethanol, 1% EmbryoMax ESCQualified Nucleosides (Millipore, Temecula, Calif.), and 10³ U/ml LIF(Millipore)] (all products were from Invitrogen, Carlsbad, Calif.,except where mentioned). On that same day, individual small moleculesfrom our known drug collection were added to the cells at a rangebetween 0.5 and 2 μM. Compound treatment was continued for 10-14 days;the cells were fixed and stained on day 14-21 using a standard ALPdetection kit (Millipore). For the second screen, 1 μM BIX was added tothe mESC medium 1 day after transduction. Five days later, in additionto 1 μM BIX, individual small molecule from the known drug collectionwas added to each well, at a range between 0.5 and 2 μM. Mouse ESC mediawith defined small molecules was refreshed every three days untilcolonies with a similar morphology to mESCs were observed, which wasusually between 14-21 days after transduction. In addition to theconfirmed compounds as indicated in the text, primary hits from thesecond synergist screen that were not further followed up also includePD173074, reversine, 5′ azacytidine, pluripotin, and dexamethasone.Further characterization studies and repeats were carried either onprimary 129S2/SvPasCrlf or ROSA26^(+/−)/OG2^(+/−) MEFs. WhenROSA26^(+/−)/OG2^(+/−) MEFs were used, the iPSC colonies could also beidentified through GFP expression, as a marker of Oct4 expression. OnceiPSC colonies were identified, they were picked for expansion on MEFfeeder cells in mESC medium. Some colonies were expanded in the presenceof the MEK inhibitor, PD0325901 at concentration of 0.5-2 μM to furtherconfirm their pluripotentiality.

Immunocytochemistry and Immunofluorescence Assay

ALP staining was performed according to manufacturer's instruction usingthe Alkaline Phosphatase Detection Kit (Millipore). Forimmunofluorescence assay, cells were fixed in 4% paraformaldehyde 15minutes at room temperature (RT) and washed with PBS. They were thenincubated in blocking buffer (BB) [0.3% Triton X-100 (Sigma-Aldrich),10% normal donkey serum (Jackson ImmunoResearch Laboratories Inc) in PBS(Invitrogen)] 30 min at RT. They were then incubated with primaryantibody overnight at 4° C. in BB. Afterward, cells were washed with PBSand incubated with secondary antibody in BB for 45-60 min at RT. Primaryantibodies were; mouse anti-Oct4 (1:200) (Santa Cruz Biotechnology,Inc., Santa Cruz, Calif.), mouse anti-SSEA1 (1:200) (Santa CruzBiotechnology Inc.), rabbit anti-Nanog (1:500) (Abcam Inc., Cambridge,Mass.), mouse anti-Sox2 (1:200) (Millipore), rabbit anti-Pdx1 (1:200) (akind gift from Dr. C. Wright), mouse anti-βIII-Tubulin (Tuj1) (1:500)(Covance Research Products Inc., Denver, Pa.), mouse anti-cardiactroponin T (CT3) (1:200) (Developmental Studies Hybridoma Bank at theUniversity of Iowa, Iowa City, Iowa), rabbit anti-albumin (DAKO).Secondary antibodies were Alexa Fluor555 donkey anti-mouse or rabbit IgG(1:500) (Invitrogen). Nuclei were detected by DAPI (Sigma-Aldrich)staining. Images were captured using a Nikon Eclipse TE2000-U/X-cite 120EXFO microscope with a photometric CoolSnap HQ2 camera.

RT-PCR Assay

RNA was extracted from iPSCs and control cell lines using the RNeasyPlus Mini Kit in combination with QIAshredder. The RNA was converted tocDNA using iScript™cDNA Synthesis Kit (BioRad, Hercules, Calif.).Amplification of specific genes was done using primers previouslypublished (Takahashi, K. et al., Cell, 131:861-872 (2007); Takahashi, K.and Yamanaka, S., Cell, 126:663-676 (2006)) and Platinum PCR SuperMix(Invitrogen) on a Mastercycler ep gradient PCR machine (Eppendorf).

Methylation Assay

DNA from R1, OG2 MEFs and OK iPSCs (passage 10) cells was isolated usingthe Non Organic DNA Isolation Kit (Millipore). The DNA was then treatedfor bisulfite sequencing with the EZ DNA Methylation-Gold Kit™ (ZymoResearch Corp., Orange, Calif.). The treated DNA was then used toamplify sequences of interest. Primers used for promoter fragmentamplification were as previously published (Blelloch, R. et al., StemCells, 24:2007-2013 (2006)). The resulting fragments were cloned usingthe TOPO TA cloning Kit for sequencing (Invitrogen) and sequencing wasdone.

Aggregation of iPSCs with Zona-Free Embryos

iPSCs were aggregated with denuded post-compacted eight-cell stageembryos to obtain aggregate chimeras. Eight-cell embryos (B6C3F1) wereflushed from females at 2.5 dpc and cultured in microdrops of KSOMmedium (10% FCS) under mineral oil. Clumps of iPSCs (10-20 cells) aftershort treatment of trypsin were chosen and transferred into microdropscontaining zona-free eight-cell embryos. Eight-cell embryos aggregatedwith iPSCs were cultured overnight at 37° C., 5% CO₂. Aggregatedblastocysts that developed from eight-cell stage were transferred intoone uterine horn of a 2.5 dpc pseudopregnant recipient. One adult malechimaera was mated with a female CD1 wild-type mouse. X-gal stainingshowed that six F1 embryos obtained from this natural mating of chimericmouse and wild-type mouse were generated by germline transmission.

Statistical Analysis

Bar graphs and statistical analyses were performed using a standardt-test on the Excel.

Microarray Analysis

OK2B iPSCs were grown in complete mES cell media on gelatin (Millipore,Temecula, Calif.) for 2 days [Knockout DMEM, 10% ES-qualified FBS, 10%Knockout serum replacement, 1% Glutamax, 1% Non-essential amino acids,penicillin/streptomycin, 0.1 mM β-mercaptoethanol, 1% EmbryoMax ESCQualified Nucleosides (Millipore), and 10³ U/ml LIF (Millipore)] (allproducts were from Invitrogen, Carlsbad, Calif., except wherementioned). RNA from duplicate wells was then isolated using RNAeasyMini Kit (Qiagen, Valencia, Calif.). Total RNA samples were amplifiedand labeled using the MessageAmp II-Biotin Enhanced Kit (Ambion, Austin,Tex.). The amplified labeled samples were then hybridized to MouseGenome 430 2.0 Arrays (Affymetrix) and analysis was performed usinghierarchical clustering (Pearson, log-transformed, row-centered values)using GenePattern (world wide web at: broad.mit.edu/cancer/software/).

Proliferation Assay

mES R1 cells were plated onto gelatin-coated 6-well plates at a densityof 2×10⁵ cells/well in complete mES cell media. Upon cell attachment,app. 12 hours, the cells were treated either with DMSO, 1 μM BIX, 2 μMBayK, and a combination of both, in triplicate. At 15, 24 and 48 hours,the cells were detached using trypsin, and counted using ahemocytometer. Trypan blue (Sigma-Aldrich, St. Louis, Mo.) was used fordead cell exclusion.

Assessment of SOX2 Expression after Compound Treatment

OG2^(+/−)/ROSA26^(+/−) MEFs were plated onto 6-well plate at a densityof 3.4×10⁴ cells per well. On the following day, the cells were treatedwith DMSO, 1 μM BIX, 2 μM BayK, and a combination of both, intriplicate, for 6 days. The media was refreshed at day 3. RNA from eachwell was then isolated using the RNAeasy Mini Kit (Quiagen). Reversetranscription of the RNA was performed using the iScript™cDNA SynthesisKit (BioRad, Hercules, Calif.). Amplification of endogenous Sox2 wasdone using primers previously published (Takahashi, K., Okita, K.,Nakagawa, M., and Yamanaka, S. (2007). Induction of pluripotent stemcells from fibroblast cultures. Nat Protoc 2, 3081-3089; Takahashi, K.,and Yamanaka, S. (2006). Induction of pluripotent stem cells from mouseembryonic and adult fibroblast cultures by defined factors. Cell 126,663-676) with Platinum PCR SuperMix (Invitrogen) on a Mastercycler® epgradient PCR machine (Eppendorf).

Example 3

This example demonstrates that incubation of mammalian cells withtranscription factor proteins is sufficient to induce pluripotency.

Gene Construction:

In order to obtain the high level protein expression in E. coli, allfour human TF gene codon region were optimized first (G A Gutman and G WHatfield (1989). PNAS. vol. 86. pp:3699-3703), and full-lengthsynthesized using DNA oligo based/PCR gene assembling technology (DaniloR Casimiro, Peter E Wright & H Jane Dyson. (1997). Structure. Vol. 5.pp: 1407-1412.). Poly-arginine tag: ESGGGGSPGRRRRRRRRRRR (SEQ ID NO:2)was added to each protein C-terminal in design (Gump J M, Dowdy S F.(2007) Trends Mol Med. Oct; 13(10):443-8). The final DNA fragment wasflanked with NdeI and XhoI site, and inserted into pET41 expressionvector NdeI-XhoI sites for protein expression. Each plasmid wereverified with DNA sequence, then transformed into BL21start competentcells for recombinant protein production using auto-induction mediumovernight (Studier F W, (2005) Protein Expr Purif. 41(1). Pp: 207-234.).

Protein Preparation

Escherichia coli BL21(DE3) cells were transformed with pET-Oct4-PTD(“PTD” refers to protein transduction domain), pET-Sox2-PTD,pET-Klf4-PTD, and pET-c-Myc-PTD separately, and the protein expressionwas done using the auto-induction method (Studier F. W., ProteinExpression and Purification, 41 (2005) 207-234.). Inclusion bodies weresolubilized and the proteins were refolded as described (LaFevre B M, WuS. & Lin X. Molecular Cancer Therapeutics 7, 1420-1429, Jun. 1, 2008.doi: 10.1158/1535-7163; Medynski D., Tuan M., Liu, W., Wu, S. & Lin, X.Protein Expression and Purification Vol. 52, 395-402, April 2007; HouW., Medynski D., Wu, S., Lin, X. & Li, L Y. Clinical Cancer ResearchVol. 11, 5595-5602, Aug. 1, 2005).

Briefly, E. coli containing an expression plasmid was inoculated into1.0 L liter of Luria-Bertani Broth containing kanamycin, induced with500 umol/L IPTG at A600 nm=0.6, and agitated for 3 hours at 37 C. Thecells were collected by centrifugation, and the pellet subjected tofreeze-and thaw cycles. The inclusion bodies released were washedextensive with a buffer containing 20 mmol/L tris, 100 mmol/L NaCl, 1%TritonX-100 (pH8.0) and dissolved in a buffer containing 8 mol/L urea,0.1 mol/L Tris, 1 mmol/L glycine, 1 mmol/L EDTA, 10 mmol/Lb-mercaptoethanol, 10 mmol/L DTT, 1 mmol/L reduced glutathione, 0.1mmol/L oxidized glutathione (pH 10) with a A280 nm=2.0. The solubilizedinclusion bodies were refolded with a rapid dilution method as described(Lin X L, Lin Y Z, Tang J., Methods Enzymol 1994, 241, 195-224; Lin X,Koelsh G., Wu. S, Downs D, Dashti A. Tang J. Proc Natl Acad Sci USA.2000; 97, 1556-1560; Kim Y T. Downs D. Wu S, et al. Eur J Biochem 2002,269: 5669-77; Michelle LaFevre-Bernt, Shili Wu, and Xinli Lin. (2008).Molecular Cancer Therapeutics. 7: pp: 1420-1429). The refolded proteinwas concentrated by N2-ultrafiltration and purified by size exclusionchromatography using Sephacryl S 300. The endotoxin concentration ineach of protein preparation was less than 100 EU/mg. Most refold proteinsamples have solubility at least >1.5 mg/ml.

Refolded proteins were concentrated using tangential flow filtration,purified using size exclusion chromatography with a Superdex-200 column(XK26×850-mm, GE, Piscataway, N.J.), and confirmed using SDS-PAGE.

Mouse fibroblasts were grown in mESC medium supplemented with 8 μg/ml ofeither Oct4/Sox2/Klf4 or Oct4/Sox2/Klf4/Myc (all proteins comprisingpoly-Arg as described above) for 6-8 hours, washed, and incubated for2-3 days in mESC media without the above-listed transcription factors.This (4-12 hours with, 1-3 days without) was repeated a number (1, 2, 3,4, or more) of times and then the cells were cultures in mESC for twoweeks. At the end of this period, the cultures were determined tocontain pluripotent cells by colony morphology and marker expression(data not shown). Notably, it was found that constant incubation of thecells with the transcription factors (i.e., without the 1-3 day periodwithout the proteins)) was toxic to the cells. While it was notnecessary, the cells were sometimes incubated with MEK inhibitor(PD0325901) and/or GSK inhibitor (CHIR99021) and/or TGFbeta inhibitor(SB431542) and the presence of these agents improved efficiency andspeed of development of pluripotent cells.

The above examples are provided to illustrate the invention but not tolimit its scope. Other variants of the invention will be readilyapparent to one of ordinary skill in the art and are encompassed by theappended claims. All publications, databases, Genbank sequences,patents, and patent applications cited herein are hereby incorporated byreference.

What is claimed is:
 1. An in vitro method of producing an inducedpluripotent stem cell from a mammalian non-pluripotent cell, the methodcomprising: (a) contacting the mammalian non-pluripotent cell with a MEKinhibitor; and (b) introducing into the mammalian non-pluripotent cell,one or more expression cassettes comprising nucleic acids encoding anOct4 polypeptide and a Klf4 polypeptide, thereby producing an inducedpluripotent stem cell; wherein the introducing step does not compriseintroducing a Myc polypeptide or introducing an expression cassettecomprising a nucleic acid encoding a Myc polypeptide.
 2. The method ofclaim 1, wherein the contacting step further comprises contacting themammalian non-pluripotent cell with a TGFβ receptor/ALK5 inhibitor. 3.The method of claim 1, wherein the contacting step further comprisescontacting the mammalian non-pluripotent cell with a TGFβ receptor/ALK5inhibitor and a GSK3 inhibitor.
 4. The method of claim 2 or 3, whereinthe TGFβ receptor/ALK5 inhibitor is selected from the group consistingof SB431542 and A-83-01.
 5. The method of claim 3, wherein the TGFβreceptor/ALK5 inhibitor is selected from the group consisting ofSB431542 and A-83-01; and the GSK3 inhibitor comprises CHIR99021.
 6. Acomposition comprising a mammalian non-pluripotent cell; one or moreexpression cassettes comprising nucleic acids encoding a Klf4polypeptide, an Oct4 polypeptide, and/or a Sox2 polypeptide; and atleast one of a TGFβ receptor/ALK5 inhibitor, a GSK3 inhibitor, and a MEKinhibitor, wherein the one or more expression cassettes does not encodea Myc polypeptide.
 7. The composition of claim 6, comprising anexpression cassette comprising a nucleic acid encoding the Oct4polypeptide.
 8. The method of claim 1, further comprising contacting themammalian non-pluripotent cell with at least one of: an agent thatinhibits H3K9 methylation or promotes H3K9 dernethylation; an L-type Cachannel agonist; an activator of the cAMP pathway; DNA methyltransferase(DNMT) inhibitor; a nuclear receptor ligand; a GSK3 inhibitor; a TGFβreceptor/ALK5 inhibitor; a HDAC inhibitor; and an Erk inhibitor.
 9. Themethod of claim 1, wherein the MEK inhibitor comprises PD0325901.